BK channel-modulating peptides and their use

ABSTRACT

The present invention concerns peptides and nucleic acids encoding the peptides, and their use for modulating large conductance Ca 2+  activated K +  (BK) channel activity in cells; for treating conditions such as presbycusis (age-related hearing loss), audiogenic seizures, alcohol addiction, cancer, and neurodegenerative disease; and for delivering a cargo moiety to the brain of a subject through the blood-brain barrier.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.15/650,375, filed Jul. 14, 2017, which claims the benefit of U.S.Provisional Application Ser. No. 62/362,316, filed Jul. 14, 2016, whichis hereby incorporated by reference herein in its entirety, includingany figures, tables, nucleic acid sequences, amino acid sequences, ordrawings.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.AA0202992 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Large conductance calcium activated channels are broadly expressed inneurons and muscle where they modulate cellular activity and function.Decades of research support an ongoing interest in modulating largeconductance Ca²⁺ activated K⁺ (BK) channel function to alter diseasestates.

Age-related hearing loss (ARHL) is a widespread medical problemaffecting approximately 30-40% of people 65 and older, while >50% ofpeople 75 and older have difficulty hearing. The primary etiology ofARHL is two-fold, i) a loss in sensitivity to sound resulting fromfunctional deficits in inner ear transduction and ii) alteration incentral auditory processing within the brain. Deficits at the inner earraise the threshold for how a loud a sound must be to be heard,particularly for higher pitched sounds. Many consonances arehigh-pitched sounds, making this deficit directly interfere with one'sability to understand speech. These types of hearing deficits can beimproved with current hearing aid technology, although some patients mayresist wearing hearing aids. Much more difficult to improve with hearingaids is the functional loss in the brain's ability to process sounds.These central auditory processing deficits make it very difficult tounderstand speech in a noisy environment.

The large conductance calcium-activated potassium (BK) channel is apromising pharmacological target. Widely expressed in human tissue whereit is gated by voltage and intracellular calcium, the BK channelregulates smooth muscle tone, endocrine secretion and neuronalexcitability. Despite broad expression, global knockout of theconstitutive pore-forming a subunit is not lethal in mice. InCaenorhabditis elegans and Drosophila, null mutations in the highlyconserved invertebrate BKα channel reduces acute ethanol intoxicationand tolerance.

Restriction of BK channel function has therapeutic value in humandisease. Human tumor growth and metastasis is supported by higher BKchannel expression and curtailed by BK channel blockers. In the CNS, aBK channel gain-of-function mutation is associated with an increasedrisk for epilepsy in humans and mice, and blocking BK channel functionsuppresses seizure activity in vivo and in vitro. BK channels areexpressed throughout the auditory system where they maintain highfrequency firing. Thus, suppression of BK channel function in theinferior colliculus (IC) may restrict audiogenic seizures, whichoriginate in this region. A reduction in high frequency firing in the ICcould also reduce tinnitus or “ringing in the ears.” Evidence fromanimal models of tinnitus suggests that a focal loss of output from thecochlea, common in many cases of tinnitus, in turn suppresses inhibitorydrive in the central auditory system resulting in hyperexcitability.This neuronal hyperexcitability is evident in both the cochlear nucleusand at the level of the IC.

A number of peptides alter BK channel function. Peptide scorpio- orconotoxins block the pore with low nanomolar affinity and highspecificity but have relatively complex structures restrictinglarge-scale synthesis. Small, endogenous peptides or peptide fragmentsmodulate BK channel function, but mainly act with lower affinity. Forexample, fragments of a BK channel auxiliary subunit (β2) inactivatecurrents in the micromolar range. There are currently no BKchannel-directed peptides for CNS pharmacological applications, havinghigh affinity and specificity, blood-brain barrier (BBB) permeabilityand amenability to large-scale synthesis.

BRIEF SUMMARY OF THE INVENTION

The present convention concerns peptides that modulate large conductanceCa²⁺ activated K⁺ (BK) channel activity, compositions containing thepeptides, and methods of using the peptides and compositions for thetreatment and prevention of diseases and disorders. In some embodiments,the peptide is one listed in Table 1 or a functional fragment or variantthereof. The peptide may be a linear peptide or a cyclic (non-linear)peptide.

In some embodiments, the peptide is LS3 (CRRGLVQVC (SEQ ID NO:3)). Thispeptide suppresses channel gating of heterologously expressed human BKchannels in the high picomolar range. In vivo experiments in wild typeor BK channel-humanized Caenorhabditis elegans show that LS3 suppresseslocomotor activity via a BK channel-specific mechanism.

The peptide can shuttle both itself and small molecule cargo across theblood-brain barrier of the mammalian brain. When tagged with fluoresceinand topically applied to the dural surface of the auditory midbrain, thepeptide traveled to a depth of at least 1200 microns into the inferiorcolliculus. Once across the blood-brain barrier, the peptide shows aconcentration-dependent effect on sound-evoked activity in inferiorcolliculus neurons. Topical application of ˜0.8 mgs/kg LS3 on the duralsurface of the mouse auditory midbrain suppressed sound evoked neuralactivity, similar to a general pore blocker, in vivo, which recoveredafter 24 hours. Similar action on sound-evoked activity was seen wheninjected systemically (i.p. at ˜0.4 mgs/kg), except that the effectstook longer to be observed. When administered at a ten-fold lower dose,the peptide lowered sound-evoked thresholds and provided betterdefinition to neuronal receptive fields in aged mice. Together thesedata show that a novel BK channel-directed peptide potently andspecifically alters channel gating and crosses the blood-brain barrierto modulate neural activity in vivo. High dose application supportsthese peptides' use for suppressing audiogenic seizures, while the lowerdose supports these peptides' use for reducing the effects ofage-related hearing loss.

One aspect of the invention concerns a peptide comprising an amino acidsequence of Table 1, i.e., selected from the group consisting of:CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ IDNO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC(SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11),CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ IDNO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC(SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19),CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ IDNO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC(SEQ ID NO:25), and CSEWPQNVC (SEQ ID NO:26), or a functional fragmentor variant of any one of the foregoing amino acid sequences. In someembodiments, the peptide is a circular peptide. In some embodiments, thepeptide has an N-terminal and C-terminal disulfide bridge (i.e., adisulfide bridge between the cysteines).

In some embodiments, the peptide comprises an amino acid sequenceselected from the group consisting of: CARGVYRVC (SEQ ID NO:1),CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ IDNO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC(SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12),CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ IDNO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC(SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20),CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ IDNO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), andCSEWPQNVC (SEQ ID NO:26). In some embodiments, the peptide is a circularpeptide. In some embodiments, the peptide has an N-terminal andC-terminal disulfide bridge (i.e., a disulfide bridge between thecysteines).

In some embodiments, the peptide consists of an amino acid sequenceselected from the group consisting of: CARGVYRVC (SEQ ID NO:1),CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ IDNO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC(SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12),CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ IDNO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC(SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20),CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ IDNO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), andCSEWPQNVC (SEQ ID NO:26). In some embodiments, the peptide is a circularpeptide. In some embodiments, the peptide has an N-terminal andC-terminal disulfide bridge (i.e., a disulfide bridge between thecysteines).

In some embodiments, the peptide comprises CRRGLVQVC (SEQ ID NO:3). Insome embodiments, the peptide comprises an amino acid sequenceconsisting of CRRGLVQVC (SEQ ID NO:3), including no further amino acidresidues. In some embodiments, the peptide consists of CRRGLVQVC (SEQ IDNO:3). In some embodiments, the peptide is a circular peptide. In someembodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines).

In some embodiments, the functional fragment or variant is a peptidecomprising an amino acid sequence comprising any one of SEQ ID NO:27-61.In some embodiments, the functional fragment or variant is a peptidecomprising an amino acid sequence consisting of any one of SEQ IDNO:27-61 (including no further amino acid residues). In someembodiments, the functional fragment or variant is a peptide consistingof an amino acid sequence consisting of any one of SEQ ID NO:27-61. Insome embodiments, the peptide is a circular peptide. In someembodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines).

Optionally, the peptide of the invention may have a heterologous aminoacid sequence or a moiety fused directly or indirectly to the peptide ofthe invention, which is then referred to as a polypeptide construct. Insome embodiments, the heterologous moiety comprises a label that isdetectable using an appropriate detection modality

In some embodiments, the polypeptide construct is a fusion polypeptidecomprising a first amino acid sequence of the invention and a secondamino acid sequence fused directly or indirectly to the first amino acidsequence. Thus, in some embodiments, the fusion polypeptide comprises afirst amino acid sequence of the invention, e.g., an amino acid sequenceselected from the group consisting of: CARGVYRVC (SEQ ID NO:1),CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ IDNO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC(SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12),CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ IDNO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC(SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20),CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ IDNO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), andCSEWPQNVC (SEQ ID NO:26), or an amino acid sequence having one or moreconservative substitutions or deletions of any one of the foregoing, anda second amino acid sequence fused directly or indirectly to the firstamino acid sequence. The second amino acid sequence may be fused to theamino-terminus (N-terminus) or carboxyl-terminus (C-terminus) of thefirst amino acid sequence. The fusion may be direct or indirect througha linker. The first amino acid sequence may be identical to or differentfrom the second amino acid sequence. The fusion polypeptide may furthercomprise one or more additional amino acid sequences directly orindirectly fused to the first amino acid sequence or second amino acidsequence, making a “multimer”.

Another aspect of the invention concerns a nucleic acid encoding any ofthe aforementioned peptides of the invention. Another aspect of theinvention concerns an expression construct, such as a viral or non-viralvector, comprising the nucleic acid encoding any of the aforementionedpeptides of the invention.

Another aspect of the invention concerns a composition comprising apeptide of the invention, a nucleic acid encoding the peptide, or anexpression construct comprising the nucleic acid; and a pharmaceuticallyacceptable carrier or diluent.

Another aspect of the invention concerns a method for treating acondition in a subject in need thereof, comprising administering anagent of the invention to the subject, wherein the condition is selectedfrom among presbycusis, audiogenic seizures, alcohol addiction, cancer,and neurodegenerative disease (e.g., Alzheimer's disease (AD),Parkinson's disease (PD), Huntington's disease). The agent may be apeptide of the invention, a nucleic acid encoding the peptide, anexpression construct comprising a nucleic acid encoding the peptide. Theagent may be administered to the subject in a composition comprising theagent and a pharmaceutically acceptable carrier or diluent.

The agent may be administered to the subject by any route effective indelivering the agent to the desired anatomical location or locations. Insome embodiments, the agent is administered systemically. In someembodiments, the agent is administered locally. In some embodiments, theagent is administered to the subject by a route selected from the groupconsisting of intravascular (e.g., intravenous or intra-arterial),intramuscular, intracutaneous, oral, intranasal, intra-ocular, topical,and transdermal.

The agent may be administered to the subject as therapy or prophylaxis.Thus, in some embodiments, the subject has the condition at the time theagent is administered, and the agent is administered to the subject astherapy for the condition. In other embodiments, the subject does nothave the condition at the time the agent is administered, and the agentis administered to prevent or delay the onset of the condition.

Optionally, one or more additional biologically active agents areadministered to the subject before, during, or after administration ofthe agent. For example, the one or more additional agents may haveactivity that is useful in treating or delaying onset of the condition.The one or more additional biologically active agents may beadministered within the same composition as the agent or in separatecomposition. Thus, the composition may include one or more additionalbiologically active agents.

In some embodiments, the agent comprises a peptide comprising orconsisting of an amino acid sequence of Table 1. In some embodiments,the agent comprises a peptide comprising CRRGLVQVC (SEQ ID NO:3). Insome embodiments, the agent comprises a peptide comprising an amino acidsequence consisting of CRRGLVQVC (SEQ ID NO:3).

Another aspect of the invention concerns a method for modulating largeconductance Ca²⁺ activated K⁺ (BK) channel activity in a cell having aBK channel in vitro or in vivo, comprising contacting the cell in vitroor in vivo with an agent of the invention. In some embodiments, the BKchannel activity is BKα channel function. The agent may be a peptide ofthe invention, a nucleic acid encoding the peptide, or an expressionconstruct comprising a nucleic acid encoding the peptide, wherein thecondition is selected from among presbycusis, audiogenic seizures,alcohol addiction, cancer, and neurodegenerative disease. The agent maybe contacted to the cell in a composition comprising the agent and apharmaceutically acceptable carrier or diluent. As used herein, the term“contacting” in this context means bringing the agent into contact withthe cell, or vice-versa, or any other manner of causing the agent andthe cell to come into contact.

In some embodiments, the agent comprises a peptide comprising orconsisting of an amino acid sequence of Table 1. In some embodiments,the agent comprises a peptide comprising CRRGLVQVC (SEQ ID NO:3). Insome embodiments, the agent comprises a peptide comprising an amino acidsequence consisting of CRRGLVQVC (SEQ ID NO:3).

In the BK modulation method, the BK channel may be native to the cell orthe BK channel may be heterologous to the cell. For example, the cellcan be genetically modified to transiently or stably express a nucleicacid encoding a BK channel that is heterologous to the cell. In someembodiments, the cell is a human cell. In other embodiments, the cell isa non-human animal cell.

Another aspect of the invention concerns a method of delivering a cargomoiety to the brain of a subject, through the blood-brain barrier (BBB),comprising administering a polypeptide construct to the subject, whereinthe polypeptide construct comprises a peptide of the inventionconjugated to the cargo moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent and Trademark Officeupon request and payment of the necessary fee.

FIG. 1. Strategy to identify peptides that affect ethanol modulation ofhuman BK channel function. Phage display was used to identify peptidesthat bind to the human BK channel. Modified bacteriophage (phagemid),each expressing one of a randomized library of small peptides, wereserially panned against ion channels expressed in HEK 293 cells.Phagemid that did not bind to hGlyRa1 or rSK2 channels but did bind tothe human BKα channel were collected, amplified and re-panned for atotal of 5 times. Phagemid were sequenced and the peptide sequencessynthesized. Peptides were screened for their ability to affect BKchannel function using C. elegans. In C. elegans, locomotion andethanol-induced locomotor suppression are BK channel-mediated processes.To test peptide function, locomotor ability with and without peptidewere compared between wild-type and BK null animals in the presence andabsence of ethanol (EtOH). For peptides suspected of modulating BKchannel function, effects on channel gating were characterized usingelectrophysiological methods. Macro and single channel currents wererecorded in patches pulled from HEK 293 cells expressing human SLOchannels in the presence and absence of peptide and ethanol.

FIGS. 2A-2B. Phagemid display selection of peptides that bind to thehuman BKα channel. 3 motifs were enriched 3000-6000 fold (p<0.0001).Positively charged amino acid clusters were enriched 100-300 fold(p<0.0001), as depicted in FIG. 2A. The latter pattern of enrichment waslikely driven by the BK channel's negatively charged outer poreresidues. A selection of representative sequences was chosen for initialtesting. These peptides included both moderately and highly enrichedmotifs. FIG. 2B depicts a sequence logo showing the relative likelihoodof amino acid expression for all twenty candidate peptides selected bythe phagemid display screen (see SEQ ID NO:1-20 of Table 1). Only theamino acid positions that were randomized in the phagemid displaylibrary (amino acids 2-7) are shown. Charged amino acids, particularlyarginine (R), were highly enriched at all positions followed by smallamino acids like glycine and alanine. Two or more amino acids in a fixedposition or three or more amino acids in a sliding position (*) thatwere shared across two or more peptides were considered motifs. Motifsare color coded by fold enrichment over theoretical frequency in theoriginal phagemid display library.

FIGS. 3A-3D. Candidate peptides showed selective BK channel functionalactivity in C. elegans. To assess the functional activity of thepeptides, worms were incubated with 750 μM peptide or vehicle (NGM) for30 minutes. Post-incubation crawl speed was measured before and after a20-minute exposure to ethanol. For all trials, peptide- andvehicle-treated worms were run in tandem. Crawl speeds were normalizedto the vehicle-treated controls in each trial. Means±SEM are shown asbars (gray or turquoise) for the peptide-treated groups and as lines forthe vehicle-treated groups (maroon). Half of the peptides causedstrongly significant (***p<0.001) changes in crawl speed for WT (N2)worms before (FIG. 3A) and/or after ethanol exposure (FIG. 3B). LS15-18showed no significant effect and were eliminated from the screen. Allother peptides were tested for effects on a strain (NM1968) expressing anull allele of the worm BK channel ortholog, slo-1. Of those thateffected WT crawl speed, only five peptides showed no effect both before(FIG. 3C) and after ethanol exposure (FIG. 3D). All peptides were testedon at least 2 days and 30 worms. For peptides exhibiting selective BKchannel functional activity (turquoise), all peptides were tested on atleast 3 days and 60 worms. Student's t-tests compared thepeptide-treated vs. vehicle-treated worms run in tandem; ***p<0.001,**p<0.01, *p<0.05.

FIGS. 4A-4B. BK channel-targeted peptides act as openers on human BKαchannels. FIGS. 4A and 4B: Pskan 1+4. Electrophysiological recordings ofhuman BKα channels expressed in HEK293 cells showed that the combinationof pskan1 and pskan4 increases channel opening. When a 10 microM peptidesolution was applied to outside-out patches held at +40 to +60 mV and700 nM to 1 μM intracellular Ca2+, the probability of opening increased1.6±0.22 fold (n=7).

FIGS. 5A-5C. Selected peptides showed functional activity in BKchannel-humanized worms. Worms expressing the ZERO isoform of the humanBKα channel on a slo-1 null background were incubated with 750 μMpeptide or vehicle (NGM) for 30 minutes. Post-incubation crawl speed wasassessed before (FIG. 5A) and after a 20-minute exposure to ethanol(FIG. 5B). For all trials, peptide- and vehicle-treated worms were runin tandem. Crawl speeds were normalized to the vehicle-treated controlsin each trial. Means±SEM are shown as bars (gray or turquoise) for thepeptide-treated groups and as lines for the vehicle-treated groups(maroon). Student's t-tests compared the peptide-treated vs.vehicle-treated worms run in tandem; ***p<0.001, *p<0.05, N>60. Fourpeptides showed significant effects (turquoise). FIG. 5C showsschematics representing the enriched amino acid motifs in the fourselect peptides. Each motif is represented by a unique color. Dualcoloring indicates the residue is shared between motifs. LS19 and LS11shared a motif, while the others expressed unique motifs.

FIGS. 6A-6H. LS3 alters the gating of human BK channels. Human BKαchannels (ZERO isoform) expressed in HEK293 cells were recorded ininside-out patches. Peptide was applied by diffusion to theextracellular side. Intracellular calcium held at 750 nM. FIGS. 6A, 6B,Single channel traces show 500 nM LS3 reduces the probability of openingat 60 mV (FIG. 6A) and 100 mV (FIG. 6B). FIGS. 6C-6E, Bar graphsdisplaying the post-peptide P_(o) relative to the starting P_(o) acrossLS3 concentrations. At 500 pM through 500 nM LS3 reduced the P_(o) ateach holding potential (pre vs. post: *p<0.05, **p<0.01, ***p<0.001,N=6-12, planned Student's t-tests). 50 pM LS3 did not significantlyalter the P_(o). FIG. 6F-6H, Bar graphs displaying the post-peptideP_(o) relative to the starting P_(o) for either 500 nM LS3 (cyclic) or500 nM of the reduced form of LS3 (linear). Unlike LS3, the linear formdid not alter P_(o) (N=6-12, planned Student's t-tests).

FIGS. 7A-7C. LS3 alters open and closed dwell times of the human BKchannel. Single human BKα channels (ZERO isoform) expressed in HEK293cells were recorded in inside-out patches. Intracellular calcium held at750 nM. FIGS. 7A-7C, Bar graphs showing one example of changes in openand closed dwell times before (red) and after (black) 500 nM LS3 (100mV). Channel open dwell times shortened (FIG. 7A). Three-componentexponential fits of closed dwell times (FIG. 7B) showed that the longestduration dwell times (component 2) lengthened while short dwell times(component 0) did not change significantly. FIG. 7C, Bar graph (right)of the change in mean open times (left) at each holding voltage showsthat LS3 shortened openings (pre vs. post mean open times: *p<0.05; N=8,planned Student's t-tests). Bar graph (left) of the change in peak timesfor short (black) and long (gray) duration closings shows that only thelongest duration closings lengthened (pre vs. post peak times: *p<0.05,**p<0.01; N=8, planned Student's t-tests).

FIGS. 8A-8F. Topical and systemic application of LS3 suppresses soundevoked activity from the mouse auditory midbrain. Baseline eRFs, frombetween 41-47 electrode sites, were typically V-shaped with varyingamounts of spontaneous activity as shown in FIGS. 8A-8C. Total spikecounts within the eRF, as denoted by the white lines, were measuredbefore and after topical application of 1 μL of a 10 μM stock of LS3 tothe surface of the IC (FIG. 8A). FIG. 8D shows that after 3 hours,driven activity fell from 430 to 130 spikes, ˜70% reduction (N=5 mice).Similarly, application of the pore blocker paxilline (1 μL of 10 μMstock) to the surface of the IC (N=5 mice) resulted in an 87% reductionin sound driven activity 5 hours post-application (FIGS. 8B and 8E).FIGS. 8C and 8F show the effects of a systemic injection (0.3 mg/kgI.P.) of LS3. A steady decline in sound evoked activity was observedover 2 hours, which accounted for an 85% reduction in spikes within theRF (N=5 mice).

FIGS. 9A-9D. LS3 alters BK channel function in wild type or humanized C.elegans. FIGS. 9A-9C, Bar graphs of crawl speeds for vehicle- (gray) andLS3-(red) treated groups show that LS3 reduced crawl speed for wild typebut not slo-1(null) worms at 75 μM (FIG. 9A), 250 μM (FIG. 9B) and 750μM (FIG. 9C; LS3 vs. vehicle for all concentrations: ***p<0.001,N=81-210, planned Student's t-tests). FIG. 9D, Bar graph of crawl speedsshows that a reduction in crawl speed by 750 μM LS3 was rescued on theslo-1 null background with extrachromosomal expression of either theworm (slo-1(+)) or the human (hslo(+)) BK channel gene (LS3 vs. vehicle:***p<0.001, N=78-162, planned Student's t-tests).

FIG. 10. Topical 1% DMSO driven spike counts taken from receptive fieldarea defined during baseline block. Topical application of 1 uL of 1%DMSO does not alter sound evoked activity from the mouse auditorymidbrain. Total spike counts (for 9 units) within the eRF were measuredbefore and up to 6 hours after topical application of DMSO. eFRAs weremeasured at 1 hour intervals and even after 6 hours, driven activityremained stable (N=1).

FIGS. 11A-11C. The 9-amino acid peptides with an N- and C-terminaldisulfide bond express motifs that were highly enriched in a screen forBK channel targeted peptides (FIG. 11A). 500 nM LS1 (SEQ ID NO:1) andLS3 (SEQ ID NO:3) suppress opening of human BKα (ZERO) expressed inHEK293 cells (FIG. 11B); all 3 peptides (LS1, LS3, and LS10) reduce meanopen times (FIG. 11C). 60 mV shown. N=6-12. Dotted line indicatesbaseline (FIGS. 11B and 11C).

FIGS. 12A, 12B-1, 12B-2, 12C, 12D, and 12E. LS10 substantially reducesthe probability of opening of the worm BK channel but not the human BKchannel. FIG. 12A shows the structure of LS10. FIGS. 12B-1 and 12B-2:representative three second traces at 100 mV before and after LS10 (500nM). Both the ZERO (FIG. 12B-1) and STREX (FIG. 12B-2) isoforms of thehuman BKα channel showed little change in the probability of opening(P_(o)). BKα channels expressed in HEK293 cells were recorded ininside-out patches. Intracellular calcium held at 750 nM. FIG. 12C showsthe mean fold change in P_(o) in response to 500 nM LS10 for the ZERO(open bars) and STREX (shaded bars) isoforms of the human BKα channel.FIG. 12D shows representative three second traces at 100 mV before andafter LS10 (500 nM). The worm BK channel (SLO-1) showed a significantreduction in the probability of opening (P_(o)). SLO-1 channelsexpressed in oocytes cells were recorded in inside-out patches.Intracellular calcium held at 5 μM. FIG. 12E shows the mean fold changein P_(o) in response to 500 nM LS10 for the SLO-1 channel.

FIGS. 13A-1, 13A-2, 13B, 13C-1, 13C-2, 13D-1, and 13D-2. LS10 shortensopen dwell times of the human BK channel. Single human BKα (ZEROisoform) channels expressed in HEK293 cells were recorded in inside-outpatches before and after 500 nM LS10. Intracellular calcium held at 750nM. FIGS. 13A-1 and 13A-2: open dwell times before (FIG. 13A-1) andafter (FIG. 13A-2) LS10 for a representative recording at 100 mV. Singleexponential fit, red line. FIG. 13B: the mean change in peak open dwelltimes in response to LS10. Paired Student's t-tests compared the pre-and post-peptide peak open durations, *p<0.05, N=5-6). FIGS. 13C-1 and13C-2: closed dwell times before (FIG. 13C-1) and after (FIG. 13C-2)LS10 for a representative recording at 100 mV. Half of the recordingsshowed a lengthening in closed times like this one. Three componentexponential fits (gray dotted-lines) and summation (red lines) alsoindicated. FIGS. 13D-1 and 13D-2: the mean change in peak short (FIG.13D-1) and long (FIG. 13D-2) duration closed dwell times in response toLS10. Paired Student's t-tests compared the pre- and post-peptide peakopen durations, *p<0.05, N=6.

FIGS. 14A-1, 14A-2, and 14B. LS10 prevents the potentiation of BKchannels by acute ethanol exposure. SLO-1 channels expressed in oocytescells were recorded in inside-out patches before. Intracellular calciumheld at 5 μM. FIGS. 14A-1 and 14A-2: representative three second tracesat 100 mV before (upper traces) and after (lower traces) bathapplication of 50 mM ethanol. Control (FIG. 14A-1) recording showsnormal potentiation of the probability of opening (P_(o)) by ethanol,which is blocked in the presence of 500 nM LS10 (FIG. 14A-2). FIG. 14B:mean P_(o) change in response to ethanol for control recordings (openbars) and in the presence of LS10 (shaded bars). Paired Student'st-tests compared the P_(o) before and after ethanol, *p<0.05, N=5.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is CARGVYRVC (lab designation: pskan1; also referred to asLS1), which is one embodiment of the peptide of the invention.

SEQ ID NO:2 is CRVAHRAVC (pskan2; also referred to as LS2), which is oneembodiment of the peptide of the invention.

SEQ ID NO:3 is CRRGLVQVC (pskan3; also referred to as LS3), which is oneembodiment of the peptide of the invention.

SEQ ID NO:4 is CPPGRGAVC (pskan4; also referred to as LS4), which is oneembodiment of the peptide of the invention.

SEQ ID NO:5 is CGMTKRPVC (pskan5; also referred to as LS5), which is oneembodiment of the peptide of the invention.

SEQ ID NO:6 is CDEMNWWVC (pskan21; also referred to as LS6), which isone embodiment of the peptide of the invention.

SEQ IDNO:7 is CERRMYRVC (pskan7; also referred to as LS7), which is oneembodiment of the peptide of the invention.

SEQ ID NO:8 is CRRAYEMVC (pskan8; also referred to as LS8), which is oneembodiment of the peptide of the invention.

SEQ ID NO:9 is CRRKRHAVC (pskan9; also referred to as LS9), which is oneembodiment of the peptide of the invention.

SEQ ID NO:10 is CAVGRLAVC (pskan10; also referred to as LS10), which isone embodiment of the peptide of the invention.

SEQ ID NO:11 is CLQEQRGVC (pskan11; also referred to as LS11), which isone embodiment of the peptide of the invention.

SEQ ID NO:12 is CRKQGRRVC (pskan12; also referred to LS12), which is oneembodiment of the peptide of the invention.

SEQ ID NO:13 is CEGRRARVC (pskan13; also referred to LS13), which is oneembodiment of the peptide of the invention.

SEQ ID NO:14 is CLDGKLDVC (pskan14; also referred to as LS14), which isone embodiment of the peptide of the invention.

SEQ ID NO:15 is CGGGGSRVC (pskan15; also referred to as LS15), which isone embodiment of the peptide of the invention.

SEQ ID NO:16 is CFTGGGGVC (pskan16; also referred to LS16), which is oneembodiment of the peptide of the invention.

SEQ ID NO:17 is CVWVKRNVC (pskan17; also referred to as LS17), which isone embodiment of the peptide of the invention.

SEQ ID NO:18 is CGMASSFVC (pskan18; also referred to as LS18), which isone embodiment of the peptide of the invention.

SEQ ID NO:19 is CDTMEQRVC (pskan19; also referred to as LS19), which isone embodiment of the peptide of the invention.

SEQ ID NO:20 is CGQQSPGVC (pskan20; also referred to s LS20), which isone embodiment of the peptide of the invention.

SEQ ID NO:21 is CWKSRWYVC (pskan6), which is one embodiment of thepeptide of the invention.

SEQ ID NO:22 is CTQAETRVC (pskan22), which is one embodiment of thepeptide of the invention.

SEQ ID NO:23 is CPKPNNTVC (pskan23), which is one embodiment of thepeptide of the invention.

SEQ ID NO:24 is CVRAPPSVC (pskan24), which is one embodiment of thepeptide of the invention.

SEQ ID NO:25 is CQAREVLVC (pskan25), which is one embodiment of thepeptide of the invention.

SEQ ID NO:26 is CSEWPQNVC (pskan26), which is one embodiment of thepeptide of the invention.

SEQ ID NO:27 is the formula CXXXXXXVC (SEQ ID NO:27), which is oneembodiment of the peptide of the invention, wherein X is any natural ornon-natural amino acid.

SEQ ID NO:28 is the enriched motif XRXXYR, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:29 is the enriched motif XRGXXX, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:30 is the enriched motif RRXXXX, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:31 is the enriched motif RXXXXA, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:32 is the enriched motif XXGRXA, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:33 is the enriched motif XXGXLX, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:34 is the enriched motif GMXXXX, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:35 is the enriched motif XXXKRX, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:36 is the enriched motif XQXXXG, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:37 is the enriched motif DXMXXX, which is one embodiment ofthe peptide of the invention, wherein X is any natural or non-naturalamino acid.

SEQ ID NO:38 is the enriched motif EQR, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:39 is the enriched motif GRR, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:40 is the enriched motif GGGG, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:41 is the formula CXRXXYRVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:42 is the formula CXRGXXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:43 is the formula CRRXXXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:44 is the formula CRXXXXAVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:45 is the formula CXXGRXAVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:46 is the formula CXXGXLXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:47 is the formula CGMXXXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:48 is the formula CXXXKRXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:49 is the formula CXQXXXGVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:50 is the formula CDXMXXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:51 is the formula CEQRXXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:52 is the formula CXEQRXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:53 is the formula CXXEQRXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:54 is the formula CXXXEQRVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:55 is the formula CGRRXXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:56 is the formula CXGRRXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:57 is the formula CXXGRRXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:58 is the formula CXXXGRRVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:59 is the formula CGGGGXXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:60 is the formula CXGGGGXVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

SEQ ID NO:61 is the formula CXXGGGGVC, which is one embodiment of thepeptide of the invention, wherein X is any natural or non-natural aminoacid.

DETAILED DESCRIPTION OF THE INVENTION

The present convention concerns peptides that modulate large conductanceCa²⁺ activated K⁺ (BK) channel activity, compositions containing thepeptides, and methods of using the peptides and compositions for thetreatment and prevention of diseases and disorders. In some embodiments,the peptide comprises, or consists of, an amino acid sequence listed inTable 1, or a functional fragment or variant thereof.

TABLE 1 Peptide sequences Peptide amino acid Name sequence Sequence IDEnriched motifs LS1 CARGVYRVC SEQ ID NO: 1 XRXXYR (SEQ ID NO: 28),(a.k.a. pskan1) XRGXXX (SEQ ID NO: 29) LS2 CRVAHRAVC SEQ ID NO: 2RXXXXA (SEQ ID NO: 31) (a.k.a. pskan2) LS3 CRRGLVQVC SEQ ID NO: 3XRGXXX (SEQ ID NO: 29), (a.k.a pskan3) RRXXXX (SEQ ID NO: 30) LS4CPPGRGAVC SEQ ID NO: 4 XXGRXA (SEQ ID NO: 32) (a.k.a. pskan4) LS5CGMTKRPVC SEQ ID NO: 5 GMXXXX (SEQ ID NO: 34), (a.k.a. pskan5)XXXKRX (SEQ ID NO: 35) LS6 CDEMNWWVC SEQ ID NO: 6 DXMXXX (SEQ ID NO: 37)(a.k.a. pskan21) LS7 CERRMYRVC SEQ ID NO: 7 XRXXYR (SEQ ID NO: 28)(a.k.a. pskan7) LS8 CRRAYEMVC SEQ ID NO: 8 RRXXXX (SEQ ID NO: 30)(a.k.a. pskan8) LS9 CRRKRHAVC SEQ ID NO: 9 RRXXXX (SEQ ID NO: 30),(a.k.a. pskan9) RXXXXA (SEQ ID NO: 31) LS10 CAVGRLAVC SEQ IDXXGRXA (SEQ ID NO: 32), (a.k.a. NO: 10 XXGXLX (SEQ ID NO: 33) pskan10)LS11 CLQEQRGVC SEQ ID EQR (SEQ ID NO: 38), (a.k.a. NO: 11XQXXXG (SEQ ID NO: 36) pskan11) LS12 CRKQGRRVC SEQ IDGRR (SEQ ID NO: 39) (a.k.a. NO: 12 pskan12) LS13 CEGRRARVC SEQ IDGRR (SEQ ID NO: 39) (a.k.a. NO: 13 pskan13) LS14 CLDGKLDVC SEQ IDXXGXLX (SEQ ID NO: 33) (a.k.a. NO: 14 pskan14) LS15 CGGGGSRVC SEQ IDGGGG (SEQ ID NO: 40) (a.k.a. NO: 15 pskan15) LS16 CFTGGGGVC SEQ IDGGGG (SEQ ID NO: 40) (a.k.a. NO: 16 pskan16) LS17 CVWVKRNVC SEQ IDXXXKRX (SEQ ID NO: 35) (a.k.a. NO: 17 pskan17) LS18 CGMASSFVC SEQ IDGMXXXX (SEQ ID NO: 34) (a.k.a. NO: 18 pskan18) LS19 CDTMEQRVC SEQ IDEQR (SEQ ID NO: 38), (a.k.a. NO: 19 DXMXXX (SEQ ID NO: 37) pskan19)LS20 (a.k.a. CGQQSPGVC SEQ ID XQXXXG (SEQ ID NO: 36) pskan20) NO: 20Pskan6 CWKSRWYVC SEQ ID NO: 21 Pskan22 CTQAETRVC SEQ ID NO: 22 Pskan23CPKPNNTVC SEQ ID NO: 23 Pskan24 CVRAPPSVC SEQ ID NO: 24 Pskan25CQAREVLVC SEQ ID NO: 25 Pskan26 CSEWPQNVC SEQ ID NO: 26In Table 1, X is any natural or non-natural amino acid.

One aspect of the invention concerns a peptide comprising an amino acidsequence of Table 1, i.e., selected from the group consisting of:CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ IDNO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQ ID NO:5), CDEMNWWVC (SEQID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC(SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11),CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ IDNO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC(SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19),CGQQSPGVC (SEQ ID NO:20), CWKSRWYVC (SEQ ID NO:21), CTQAETRVC (SEQ IDNO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC(SEQ ID NO:25), and CSEWPQNVC (SEQ ID NO:26), or a functional fragmentor variant of any one of the foregoing amino acid sequences.

The peptides of the invention may be a linear peptide, or a cyclic(non-linear) peptide. Cyclic peptides are polypeptide chains in whichthe amino termini and carboxyl termini; amino termini and side chain;carboxyl termini and side chain; or side chain and side chain are linked(e.g., with a covalent bond) that generates the ring (see Joo S, “CyclicPeptides as Therapeutic Agents and Biochemical Tools,” Biomol Ther(Seoul), 2012 January; 20(1): 19-26, which is incorporated herein byreference). Cyclic peptides can generally be classified according to thetypes of bonds that comprise the ring: homodetic cyclic peptides, cyclicisopeptides, cyclic depsipeptides, and bicyclic peptides. Homodeticcyclic peptides, such as cyclosporine A, are those in which the ring iscomposed exclusively of normal peptide bonds (i.e., between the alphacarboxyl of one residue to the alpha amine of another). Cyclicisopeptides contain at least one non-alpha amide linkage, such as alinkage between the side chain of one residue to the alpha carboxylgroup of another residue, as in microcystin and bacitracin. Cyclicdepsipeptides, such as aureobasidin A and HUN-7293, have at least onelactone (ester) linkage in place of one of the amides. Some cyclicdepsipeptides are cyclized between the C-terminal carboxyl and the sidechain of a Thr or Ser residue in the chain, such as kahalalide F,theonellapeptolide, and didemnin B. Bicyclic peptides such as theamatoxin amanitin and the phallotoxin phalloidin contain a bridginggroup, generally between two of the side chains. In the amatoxins, thisbridge is formed as a thioether between the Trp and Cys residues. Otherbicyclic peptides include echinomycin, triostin A, and Celogentin C.There are a number of cyclic peptide hormones which are cyclized througha disulfide bond between two cysteines, as in somatostatin and oxytocin.

Depending on the cyclization position, there are several methods tosynthesize cyclic peptides: head-to-tail, side-chain-to-side-chain,head-to-side-chain, and side-chain-to-tail (see figure below). Whilehead-to-tail cycles are usually formed by amide bond formation,side-chain-to-side-chain cycles are most often synthesized via Cys-Cysor amide bond formation.

The peptide disulfide bridge strategy readily synthesizes two thiol (SH)groups from the side chain of cysteine or cysteine analogues. Thisstrategy enables either specific intra- or intermolecular oxidationusing appropriate protecting group chemistry to avoid undesired linkage.The reaction can be followed by HPLC and MALDI TOF mass spectrometrywith the linear peptide losing two mass units (2H) on cyclization.

In general, a disulfide bridge can be formed intermolecularly (twopeptide molecules are linked via the disulfide bridge), resulting ineither: homodimers (two identical peptides) or heterodimers (twodifferent peptides); or intramolecularly (cyclization within one peptidemolecule). While head-to-tail cyclization is usually formed by using anamide bond formation, side-chain-to-side-chain peptide cyclization ismost often synthesized via Cys-Cys or amide bond formation. Peptideswith two or more disulfide bridges require selective protection of thecysteine side chains to ensure that the correct disulfide bridges areformed. Peptides with up to 4 disulfide bonds in one peptide can beproduced using site-specific orthogonal chemistry or thermodynamicstability methods. Contact us to discuss your project details.

Cyclic peptides can also be synthesized by linking the N-terminus of thepeptide to the C-terminus via an amide bond. The amino side chains ofLys and Orn and the carboxyl side chains of Asp and Glu can also be usedto construct cyclic peptides via an amide bond. Amide bond is morechemically stable than disulfide bridge. Depending on functional groupsof a peptide, cyclic peptide synthesis can be formed in four differentways: head-to-tail between N-terminus and C-terminus; head-to-side chainbetween N-terminus and an internal COOH (e.g., the β-COOH-group of Aspor γ-COOH-group of Glu); side chain-to-tail between internal NH2s andC-terminus (e.g., the ε-NH2-group of Lys); and side-chain-to-side-chainbetween an internal NH2 and an internal COOH (e.g., the ε-NH2-group ofLys with either the β-COOH-group of Asp or γ-COOH-group of Glu). Stapledpeptide synthesis and click chemistry may also be used for cyclicpeptide production.

In some embodiments, the peptide has an N-terminal and C-terminaldisulfide bridge (i.e., a disulfide bridge between the cysteines).

In some embodiments, the peptide comprises an amino acid sequenceselected from among: CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ ID NO:2),CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQ IDNO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC(SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13).CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ IDNO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC(SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC (SEQ ID NO:21),CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ IDNO:24), CQAREVLVC (SEQ ID NO:25), and CSEWPQNVC (SEQ ID NO:26). In someembodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines).

In some embodiments, the peptide consists of an amino acid sequenceselected from the group consisting of: CARGVYRVC (SEQ ID NO:1),CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ IDNO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC(SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12),CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ IDNO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC(SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20),CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ IDNO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), andCSEWPQNVC (SEQ ID NO:26). In some embodiments, the peptide has anN-terminal and C-terminal disulfide bridge (i.e., a disulfide bridgebetween the cysteines).

Several motifs have been identified among the peptides. In someembodiments, the peptide comprises an amino acid sequence comprising theformula CXXXXXXVC (SEQ ID NO:27), wherein X is any natural ornon-natural amino acid, and having an N-terminal and C-terminaldisulfide bridge (i.e., a disulfide bridge between the cysteines). Insome embodiments, the peptide comprises an amino acid sequenceconsisting of the formula CXXXXXXVC (SEQ ID NO:27), wherein X is anynatural or non-natural amino acid. In some embodiments, the peptide hasan N-terminal and C-terminal disulfide bridge (i.e., a disulfide bridgebetween the cysteines).

Enriched motifs are listed in Table 1. Furthermore, the followingthirteen enriched motifs have been identified: XRXXYR (SEQ ID NO:28),XRGXXX (SEQ ID NO:29), RRXXXX (SEQ ID NO:30), RXXXXA (SEQ ID NO:31),XXGRXA (SEQ ID NO:32), XXGXLX (SEQ ID NO: 33), GMXXXX (SEQ ID NO:34),XXXKRX (SEQ ID NO:35), XQXXXG (SEQ ID NO:36), DXMXXX (SEQ ID NO:37), EQR(SEQ ID NO:38), GRR (SEQ ID NO:39), and GGGG (SEQ ID NO:40), wherein Xis any natural or non-natural amino acid, and those motifs without an“X” can slide anywhere en bloc within the six variable amino acidpositions of CXXXXXXVC (SEQ ID NO:27). Thus, in some embodiments, thepeptide comprises an amino acid sequence comprising or consisting of:CXRXXYRVC (SEQ ID NO:41), CXRGXXXVC (SEQ ID NO:42), CRRXXXXVC (SEQ IDNO:43), CRXXXXAVC (SEQ ID NO:44), CXXGRXAVC (SEQ ID NO:45), CXXGXLXVC(SEQ ID NO:46), CGMXXXXVC (SEQ ID NO:47), CXXXKRXVC (SEQ ID NO:48),CXQXXXGVC (SEQ ID NO:49), CDXMXXXVC (SEQ ID NO:50), CEQRXXXVC (SEQ IDNO:51), CXEQRXXVC (SEQ ID NO:52), CXXEQRXVC (SEQ ID NO:53), CXXXEQRVC(SEQ ID NO:54), CGRRXXXVC (SEQ ID NO:55), CXGRRXXVC (SEQ ID NO:56),CXXGRRXVC (SEQ ID NO:57), CXXXGRRVC (SEQ ID NO:58), CGGGGXXVC (SEQ IDNO:59), CXGGGGXVC (SEQ ID NO:60), or CXXGGGGVC (SEQ ID NO:61), wherein Xis any natural or non-natural amino acid. In some embodiments, thepeptide has an N-terminal and C-terminal disulfide bridge (i.e., adisulfide bridge between the cysteines).

In some embodiments, the peptide comprises an amino acid sequencecomprising, or consisting of, an enriched motif of Table 1, i.e., SEQ IDNO:28-40, wherein X is any natural or non-natural amino acid.

In some embodiments, the peptide comprises CRRGLVQVC (SEQ ID NO:3). Insome embodiments, the peptide comprises an amino acid sequenceconsisting of CRRGLVQVC (SEQ ID NO:3). In some embodiments, the peptidehas an N-terminal and C-terminal disulfide bridge (i.e., a disulfidebridge between the cysteines).

In some embodiments, the peptide is in isolated or purified form.

Peptides that comprise an indicated amino acid sequence (e.g., bynumeric identifier) may further comprise additional moieties attacheddirectly or indirectly to the amino acid sequence, such as additionalamino acids, nucleic acids, or small molecules, for example. Peptidesthat comprise an amino acid sequence consisting of an indicated sequence(e.g., by numeric identifier) include no additional amino acids as partof that amino acid sequence, but may include other moieties attacheddirectly or indirectly to the amino acid sequence, such as nucleic acidsor small molecules, for example.

Optionally, the peptide of the invention may have a heterologous aminoacid sequence or a moiety fused or otherwise coupled directly orindirectly to any portion of the amino acid sequence, which is thenreferred to as a polypeptide construct. In some embodiments, theheterologous moiety comprises a label that is detectable using anappropriate detection modality. For example, the detectable label may bea chemiluminescent structural element, a radioactive isotope, or anenzyme to generate a color reaction. The moiety fused to the amino acidsequence may be a nucleic acid such as a DNA or RNA molecule.Polypeptide constructs may be linear or cyclic.

The peptides of the invention may comprise chemical modifications in theside chain or at the N- and/or C-terminal for improving biological orchemical properties such as bio availability, stability, effectivity.The modification may also provide for a detectable label, for example achem-iluminescent structural element, one or more radioactive isotopesin one or more side chains of an amino acid in the peptide, an enzymewhich is able to generate a color reaction and the like.

A large number of fluorescent and chemiluminescent compounds have beenshown to be useful for labeling proteins and nucleic acids. Examples ofcompounds that may be used as the dye portion can include but are notlimited to xanthene, anthracene, cyanine, porphyrin and coumarin dyes.Examples of xanthene dyes that may be coupled to the peptides caninclude but are not limited to fluorescein, 6-carboxyfluorescein(6-FAM), 5-carboxyfluorescein (5-Fam), 5- or6-carboxy-4,7,2′,7′-tetrachlorofluorescein (TET), 5- or6-carboxy-4′5′2′4′5′7′ hexachlorofluorescein (HEX), 5′ or6′-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE) rhodol, rhodamine,tetramethylrhodamine (TAMRA), 4,7-dichlorotetramethyl rhodamine(DTAMRA), rhodamine X (ROX) and Texas Red. Examples of cyanine dyes thatmay find use with the peptides include but are not limited to Cy 3, Cy3.5, Cy 5, Cy 5.5, Cy 7 and Cy 7.5. Other dyes that may find use withthe peptides can include but are not limited to energy transfer dyes,composite dyes and other aromatic compounds that give fluorescentsignals. Chemiluminescent compounds that may be used with the peptidesinclude but are not limited to dioxetane and acridinium esters. Itshould also be understood that ligands and dyes are not mutuallyexclusive groups. For instance, fluorescein is a well known example of amoiety that has been used as a fluorescent label and also as an antigenfor labeled antibodies.

The heterologous amino acid sequence or moiety may have a physiologicalfunction such as an antibody or antibody fragment, scaffolds such aslipocalin, ankyrin, fibronectin, transferrin, tetranectin, adnectin,albumin, uteroglobin, or protein A, functional peptides such astransferrin, peptides useful for diagnostic applications, fluorescentpolypeptide such as green fluorescent protein (GFP), or peptide tagsenabling immobilization on technical surfaces, such as hexahistidine, orglutathione-S-transferase (GST).

In some embodiments, the polypeptide construct is a fusion polypeptidecomprising a first amino acid sequence of the invention and a secondamino acid sequence fused directly or indirectly to the first amino acidsequence. Thus, in some embodiments, the fusion polypeptide comprises afirst amino acid sequence of the invention, e.g., an amino acid sequenceselected from the group consisting of: CARGVYRVC (SEQ ID NO:1),CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ IDNO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC(SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12),CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ IDNO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC(SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20),CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ IDNO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), andCSEWPQNVC (SEQ ID NO:26), or an amino acid sequence having one or moreconservative substitutions or deletions of any one of the foregoing, anda second amino acid sequence fused directly or indirectly to the firstamino acid sequence. In some embodiments, the peptide has an N-terminaland C-terminal disulfide bridge (i.e., a disulfide bridge between thecysteines). The second amino acid sequence may be fused to theamino-terminus (N-terminus) or carboxyl-terminus (C-terminus) of thefirst amino acid sequence. The fusion may be direct or indirect througha linker (e.g., a chemical element linker or amino acid linker). Thefirst amino acid sequence may be identical to or different from thesecond amino acid sequence. The fusion polypeptide may further compriseone or more additional amino acid sequences directly or indirectly fusedto the first amino acid sequence or second amino acid sequence, making a“multimer”.

The peptides of the invention can shuttle themselves and cargo moleculesacross the blood-brain barrier of the mammalian brain. Another aspect ofthe invention concerns a method of delivering a cargo moiety to thebrain of a subject, through the blood-brain barrier (BBB), comprisingadministering a polypeptide construct described above to the subject,wherein the polypeptide construct comprises a peptide of the inventionconjugated to the cargo moiety. The polypeptide construct can beadministered to the subject by any method outside the brain, allowingthe polypeptide construct bearing one or more cargo moieties to beported into the brain of the subject through the BBB. In someembodiments, the polypeptide construct is administered intravascularly(e.g., intravenously or intra-arterially). In the delivery method, thecargo moiety can be any moiety (inclusive of moieties) that may becoupled to the peptide and not prevent the peptide from passing throughthe BBB. For example, the cargo moiety to be ported across the BBB maybe a small molecule(s), amino acid(s), nucleic acid(s) such as DNA orRNA, detectable label(s), etc.

Examples of detectable labels that may be coupled to the peptides of theinvention include, for example, fluorescent labels, chemiluminescentlabels, and bioluminescent labels. Fluorescent labeling compounds thatmay be used are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine. Thepeptide also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedpeptide can then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester. Likewise, a bioluminescent compound can be used to labelthe peptide of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Typical bioluminescent compounds useful forlabeling are luciferin, luciferase and aequorin.

The peptides may be produced recombinantly or synthesized. Recombinantlyexpressed peptides can be purified using any one of several methodsreadily known in the art, including ion exchange chromatography,hydrophobic interaction chromatography, affinity chromatography, gelfiltration, and reverse phase chromatography. The peptide is preferablyproduced in purified form (preferably at least about 80% or 85% pure,more preferably at least about 90% or 95% pure) by conventionaltechniques. Depending on whether the recombinant host cell is made tosecrete the polypeptide into growth medium (see U.S. Pat. No. 6,596,509to Bauer et al., which is hereby incorporated by reference in itsentirety), the peptides can be isolated and purified by centrifugation(to separate cellular components from supernatant containing thesecreted peptide) followed by sequential ammonium sulfate precipitationof the supernatant. The fraction containing the peptide is subjected togel filtration in an appropriately sized dextran or polyacrylamidecolumn to separate the peptides from other proteins. If necessary, thepeptide fraction may be further purified by HPLC.

Peptides (at least those containing peptide linkages between amino acidresidues) may be synthesized by the Fmoc-polyamide mode of solid-phasepeptide synthesis as disclosed by Lu et al. (1981) J. Org. Chem. 46,3433-3436, and references therein. Temporary N-amino group protection isafforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitivecleavage of this highly base-labile protecting group is achieved byusing 20% piperidine in N,N-dimethylformamide. Side-chainfunctionalities may be protected as their butyl ethers (in the case ofserine threonine and tyrosine), butyl esters (in the case of glutamicacid and aspartic acid), butyloxycarbonyl derivative (in the case oflysine and histidine), trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support can be based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethylphenoxyacetic acid derivative. All amino acidderivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversedN,N-dicyclohexyl-carbodiimide/lhydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used are ethanedithiol, phenol, anisole and water,the exact choice depending on the constituent amino acids of the peptidebeing synthesized.

A “peptide” refers to a chain of two or more amino acids of any length.In some embodiments, the peptides of the invention have an overalllength of between 5 and 100 amino acids, between 5 and 30 amino acids,between 5 and 12 amino acids, between 8 and 10 amino acids, or 9 aminoacids. Multimers can have lengths that are multiples of these ranges. Apeptide may be linear or circular (non-linear). In some embodiments, thepeptide has an N-terminal and C-terminal disulfide bridge (i.e., adisulfide bridge between the cysteines).

Another aspect of the invention concerns a nucleic acid encoding any ofthe aforementioned peptides of the invention. Another aspect of theinvention concerns an expression construct, such as a viral or non-viralvector, comprising the nucleic acid encoding any of the aforementionedpeptides of the invention.

Another aspect of the invention concerns a composition comprising apeptide of the invention, a nucleic acid encoding the peptide, or anexpression construct comprising the nucleic acid or expressionconstruct; and a pharmaceutically acceptable carrier or diluent.

Another aspect of the invention concerns a method for treating acondition in a subject in need thereof, comprising administering anagent of the invention to the subject, wherein the condition is selectedfrom among presbycusis, audiogenic seizures, alcohol addiction, cancer,and neurodegenerative disease (e.g., Alzheimer's disease (AD),Parkinson's disease (PD), Huntington's disease). The agent may be apeptide of the invention, a nucleic acid encoding the peptide, anexpression construct comprising a nucleic acid encoding the peptide. Theagent may be administered to the subject in a composition comprising theagent and a pharmaceutically acceptable carrier or diluent.

The agent may be administered to the subject by any route effective indelivering the agent to the desired anatomical location or locations. Insome embodiments, the agent is administered systemically. In someembodiments, the agent is administered locally. In some embodiments, theagent is administered to the subject by a route selected from the groupconsisting of intravascular (e.g., intravenous or intra-arterial),intramuscular, intracutaneous, oral, intranasal, intra-ocular, topical,and transdermal.

The agent may be administered to the subject as therapy or prophylaxis.Thus, in some embodiments, the subject has the condition at the time theagent is administered, and the agent is administered to the subject astherapy for the condition. In other embodiments, the subject does nothave the condition at the time the agent is administered, and the agentis administered to prevent or delay the onset of the condition.

Optionally, one or more additional biologically active agents areadministered to the subject before, during, or after administration ofthe agent. For example, the one or more additional agents may haveactivity that is useful in treating or delaying onset of the condition.The one or more additional biologically active agents may beadministered within the same composition as the agent or in separatecomposition. Thus, the composition may include one or more additionalbiologically active agents.

In some embodiments, the agent comprises a peptide of Table 1. In someembodiments, the peptide of Table 1 has an N-terminal and C-terminaldisulfide bridge (i.e., a disulfide bridge between the cysteines). Insome embodiments, the agent comprises a peptide comprising CRRGLVQVC(SEQ ID NO:3). In some embodiments, the agent comprises a peptidecomprising an amino acid sequence consisting of CRRGLVQVC (SEQ ID NO:3).

Another aspect of the invention concerns a method for modulating BKchannel activity in a cell having a BK channel in vitro or in vivo,comprising contacting the cell in vitro or in vivo with an agent of theinvention. In some embodiments, the BK channel activity is BKα channelfunction (see, for example, Lee U S and J Cui, “BK channel activation:structural and functional insights, Trends Neurosci, 2010,33(9):415-423, which is incorporated herein by reference). In someembodiments, the peptide suppresses BK channel via modulation of thealpha subunit activity, rather than blocking the pore to prevent ionflow.

The agent may be a peptide of the invention, a nucleic acid encoding thepeptide, or an expression construct comprising a nucleic acid encodingthe peptide, wherein the condition is selected from among presbycusis,audiogenic seizures, alcohol addiction, cancer, and neurodegenerativedisease. The agent may be contacted to the cell in a compositioncomprising the agent and a pharmaceutically acceptable carrier ordiluent. Optionally, the composition may include other biologicallyactive agents. As used herein, the term “contacting” in this contextmeans bringing the agent into contact with the cell, or vice-versa, orany other manner of causing the agent and the cell to come into contact.

In some embodiments, the agent comprises a peptide of Table 1. In someembodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines). In someembodiments, the agent comprises a peptide comprising CRRGLVQVC (SEQ IDNO:3). In some embodiments, the agent comprises a peptide comprising anamino acid sequence consisting of CRRGLVQVC (SEQ ID NO:3).

In the BK modulation method, the BK channel may be native to the cell orthe BK channel may be heterologous to the cell. For example, the cellcan be genetically modified to transiently or stably express a nucleicacid encoding a BK channel that is heterologous to the cell. In someembodiments, the cell is a human cell. In other embodiments, the cell isa non-human animal cell.

The invention also concerns cells comprising a nucleic acid encoding apeptide of the invention. The peptide may be native to the cell or thepeptide may be heterologous to the cell. For example, the cell can begenetically modified to transiently or stably express a nucleic acidencoding a peptide. In some embodiments, the cell is a human cell. Inother embodiments, the cell is a non-human animal cell. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellis in isolated or purified form.

The peptides of the subject invention encompass those specificallyexemplified herein, as well as any fragments and variants thereof thatretain the desired biological activity. Typically, the desiredbiological activity will be modulation of BK channel function. In someembodiments, the BK channel activity is BKα channel function. In someembodiments, the peptide activity is suppression of the BK channel viamodulation of the alpha subunit activity, as opposed to blocking thepore to prevent ion flow.

The peptides contemplated in the subject invention include the specificpeptides exemplified herein as well as equivalent peptides which may be,for example, somewhat longer or shorter than the peptides exemplifiedherein. For example, using the teachings provided herein, a personskilled in the art could readily make peptides having from 1 to about 5,10, 15, 20, 25, 30, 35, 40, 50, 60, 70 or more amino acids added to, orremoved from, either end of the disclosed peptides using standardtechniques known in the art. In one embodiment, amino acids are removedfrom the N-terminus of a peptide of the invention. In a specificembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 50, 60, 70 or more amino acids can, independently,be removed from either or both ends of a peptide of the invention. Theskilled artisan, having the benefit of the teachings disclosed in thesubject application, can determine whether a variant peptide retains thebiological activity of the specific peptides exemplified herein.

Also within the scope of the subject invention are peptides which havethe same amino acid sequences of a peptide exemplified herein except foramino acid substitutions, additions, or deletions within the sequence ofthe peptide, as long as these variant peptides retain substantially thesame relevant biological activity as the peptides specificallyexemplified herein. For example, conservative amino acid substitutionswithin a peptide which do not affect the ability of the peptide to, forexample, modulation BK channel function would be within the scope of thesubject invention. Thus, the peptides disclosed herein should beunderstood to include variants and fragments, as discussed above, of thespecifically exemplified sequences.

The subject invention further includes nucleic acids (also referred toherein as polynucleotides) comprising nucleotide sequences which encodethe peptides disclosed herein. In one embodiment, a polynucleotidecomprises a nucleotide sequence which encodes a peptide comprising oneor more of the amino acid sequences of Table 1, or a functional fragmentor variant of the peptide. These nucleotide sequences can be readilyconstructed by those skilled in the art having the knowledge of theprotein and peptide amino acid sequences which are presented herein. Aswould be appreciated by one skilled in the art, the degeneracy of thegenetic code enables the artisan to construct a variety of nucleotidesequences that encode a particular peptide or protein. The choice of aparticular nucleotide sequence could depend, for example, upon the codonusage of a particular expression system.

The subject invention contemplates the use of the peptides describedherein in pharmaceutical compositions for administration to an animal orhuman subject for the treatment of conditions selected from amongpresbycusis, audiogenic seizures, alcohol addiction, cancer, andneurodegenerative disease. The peptides of the subject invention can beprepared in pharmaceutically acceptable carriers or diluents foradministration to humans or animals in a physiologically tolerable form.Materials and methods for preparing such compositions are known in theart.

The peptides of the subject invention can be administered using avariety of techniques that are known in the art. In one embodiment, oneor more peptides of the invention are administered as a topicalpreparation to the skin or an external membrane of a person or animal.The peptides can be encapsulated in liposomes that are targeted tospecific cells or tissues and the liposome-encapsulated peptidesdelivered to the cells or tissue either in vitro, in vivo, or ex vivo.Procedures for preparing liposomes and encapsulating compounds withinthe liposome are well known in the art. See, for example, U.S. Pat. No.5,252,348, which issued to Schreier et al. Peptides can also beconjugated or attached to other molecules, such as an antibody, thattargeted a specific cell or tissue. Peptides can also be administeredusing a drug delivery system similar to that described in U.S. Pat. No.4,625,014, which issued to Senter et al.

A further aspect of the claimed invention is the use of the claimedpeptides to produce antibodies, both polyclonal and monoclonal. Theseantibodies can be produced using standard procedures well known to thoseskilled in the art. These antibodies may be used as diagnostic andtherapeutic reagents. For example, interfering antibodies that bind tothe peptide can be used as an antagonist to block the function of thepeptide. Antibodies that are reactive with the peptides of the subjectinvention can also be used to purify the peptides from a crude mixture.

An antibody that is contemplated by the present invention can be in anyof a variety of forms, including a whole immunoglobulin, an antibodyfragment such as Fv, Fab, and similar fragments, as well as a singlechain antibody that includes the variable domain complementaritydetermining regions (CDR), and similar forms, all of which fall underthe broad term “antibody,” as used herein.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment of an antibody yields an F(ab′)₂fragment that has two antigen binding fragments, which are capable ofcross-linking antigen, and a residual other fragment (which is termedpFc′). Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “antigen binding fragment” withrespect to antibodies, refers to, for example, Fv, F(ab) and F(ab′)₂fragments.

Antibody fragments can retain an ability to selectively bind with theantigen or analyte are contemplated within the scope of the inventionand include:

(1) Fab is the fragment of an antibody that contains a monovalentantigen-binding fragment of an antibody molecule. A Fab fragment can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain.

(2) Fab′ is the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain. Two Fab′ fragmentsare obtained per antibody molecule. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region.

(3) (Fab′)₂ is the fragment of an antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction. F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)—V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen-bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain (V_(L)), thevariable region of the heavy chain (V_(H)), linked by a suitablepolypeptide linker as a genetically fused single chain molecule. Suchsingle chain antibodies are also referred to as “single-chain Fv” or“sFv” antibody fragments. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv fragments, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, N.Y.,pp. 269 315 (1994).

Antibodies within the scope of the invention can be of any isotype,including IgG, IgA, IgE, IgD, and IgM. IgG isotype antibodies can befurther subdivided into IgG1, IgG2, IgG3, and IgG4 subtypes. IgAantibodies can be further subdivided into IgA1 and IgA2 subtypes.

Antibodies of the subject invention can be genus or species specific toa target. Antibodies of the invention can be prepared using standardtechniques known in the art. Antibodies useful in the invention can bepolyclonal or monoclonal antibodies. Monoclonal antibodies can beprepared using standard methods known in the art (Kohler et al., 1975).Antibodies of the invention can be mammalian antibodies, includingmouse, rat, goat, rabbit, pig, dog, cat, monkey, chimpanzee, ape, orhuman.

The subject invention also concerns compositions comprising one or morepeptides or polynucleotides of the invention. In one embodiment, acomposition further comprises a suitable carrier, diluent, or buffer.Compositions contemplated within the scope of the invention can compriseone or more peptides or polynucleotides of the invention and,optionally, one or more other compounds for treating the condition to betreated, e.g., presbycusis, audiogenic seizures, alcohol addiction,cancer, and neurodegenerative disease. In one embodiment, thecomposition comprises a peptide or polynucleotide of the invention in apharmaceutically or physiologically acceptable carrier, buffer, ordiluent.

In one embodiment, peptides, polynucleotides, antibodies, and otheragents of the invention are modified so as to enhance uptake into acell. In one embodiment, a lipophilic group is attached to a peptide,polynucleotide, or other agent of the invention. In one embodiment, apalmitic acid is attached to a peptide of the invention. In a specificembodiment, a palmitoyl-lysine group is attached to the peptide, forexample at the N-terminus of the peptide. Other methods for enhancinguptake of a peptide, polynucleotide, and antibody into a cell are knownin the art and are contemplated within the scope of the invention.

Peptides, polynucleotides, antibodies, compositions, and other agents ofthe invention can also be delivered into cells by encapsulation of thepeptide, polynucleotide, antibody, and other agents of the inventionwithin a liposome. Methods for encapsulation of peptides,polynucleotides, antibodies, and other agents of the invention withinliposomes are well known in the art.

Peptides having substitution of amino acids other than thosespecifically exemplified in the subject peptides are also contemplatedwithin the scope of the present invention. For example, non-naturalamino acids can be substituted for the amino acids of a peptide of theinvention, so long as the peptide having substituted amino acids retainssubstantially the same activity as the peptide in which amino acids havenot been substituted. Examples of non-natural amino acids include, butare not limited to, ornithine, citrulline, hydroxyproline, homoserine,phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, γ-aminobutyric acid, ε-amino hexanoic acid, 6-amino hexanoic acid, 2-aminoisobutyric acid, 3-amino propionic acid, norleucine, norvaline,sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, C-methyl amino acids,N-methyl amino acids, and amino acid analogues in general. Non-naturalamino acids also include amino acids having derivatized side groups.Furthermore, any of the amino acids in the protein can be of the D(dextrorotary) form or L (levorotary) form.

Amino acids can be generally categorized in the following classes:non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby a peptide having an amino acid of one class isreplaced with another amino acid of the same class fall within the scopeof the subject invention so long as the peptide having the substitutionstill retains substantially the same biological activity as a peptidethat does not have the substitution. Table 2 below provides a listing ofexamples of amino acids belonging to each class.

TABLE 2 Class of Amino Acid Examples of Amino Acids NonpolarAla, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged PolarGly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

Single letter amino acid abbreviations are defined in Table 3.

TABLE 3 Letter Symbol Amino Acid A Alanine B Asparagine or aspartic acidC Cysteine D Aspartic Acid E Glutamic Acid F Phenylalanine G Glycine HHistidine I Isoleucine K Lysine L Leucine M Methionine N Asparagine PProline Q Glutamine R Arginine S Serine T Threonine V Valine WTryptophan Y Tyrosine Z Glutamine or glutamic acid

The peptides of the present invention can be formulated intopharmaceutically-acceptable salt forms. Pharmaceutically-acceptablesalts of the peptides of the invention can be prepared usingconventional techniques. “Pharmaceutically acceptable salt” includesboth acid and base addition salts. A pharmaceutically acceptable salt ofany one of the peptides described herein is intended to encompass anyand all pharmaceutically suitable salt forms. Preferred pharmaceuticallyacceptable salts described herein are pharmaceutically acceptable acidaddition salts and pharmaceutically acceptable base addition salts. Insome embodiments, the pharmaceutically acceptable salt comprisesacetate, chloride, or trifluoroacetic acid (TFA) salt.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid,hydrofluoric acid, phosphorous acid, and the like. Also included aresalts that are formed with organic acids such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, alkanedioic acids, aromatic acids, aliphatic and. aromaticsulfonic acids, etc. and include, for example, acetic acid,trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Exemplary salts thus include sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates,trifluoroacetates, propionates, caprylates, isobutyrates, oxalates,malonates, succinate suberates, sebacates, fumarates, maleates,mandelates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates,phenylacetates, citrates, lactates, malates, tartrates,methanesulfonates, and the like. Also contemplated are salts of aminoacids, such as arginates, gluconates, and galacturonates (see, forexample, Berge S. M. et al., “Pharmaceutical Salts,” Journal ofPharmaceutical Science, 66:1-19 (1997), which is hereby incorporated byreference in its entirety). Acid addition salts of basic compounds maybe prepared by contacting the free base forms with a sufficient amountof the desired acid to produce the salt according to methods andtechniques with which a skilled artisan is familiar.

“Pharmaceutically acceptable base addition salt” refers to those saltsthat retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Pharmaceutically acceptable base addition salts may beformed with metals or amines, such as alkali and alkaline earth metalsor organic amines. Salts derived from inorganic bases include, but arenot limited to, sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Salts derived from organic bases include, but are not limited to, saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, for example, isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, ethanolamine,diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,N,N-dibenzylethylenediamine, chloroprocaine, hydrabamine, choline,betaine, ethylenediamine, ethylenedianiline, N-methylglucamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like. See Bergeet al., supra.

The subject invention also concerns polynucleotide expression constructsthat comprise a nucleic acid of the present invention comprising anucleotide sequence encoding a peptide of the present invention. In oneembodiment, the polynucleotide encodes a peptide comprising the aminoacid sequence CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ ID NO:2),CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQ IDNO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC(SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13).CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ IDNO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC(SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC (SEQ ID NO:21),CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ IDNO:24), CQAREVLVC (SEQ ID NO:25), CSEWPQNVC (SEQ ID NO:26), or afragment or variant thereof that exhibits substantially the sameactivity as the full-length non-variant peptide. In some embodiments,the peptide is a circular peptide. In some embodiments, the peptide hasan N-terminal and C-terminal disulfide bridge (i.e., a disulfide bridgebetween the cysteines).

In one embodiment, the polynucleotide encodes a peptide comprising anamino acid sequence consisting of CARGVYRVC (SEQ ID NO:1), CRVAHRAVC(SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ ID NO:4),CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQ IDNO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC (SEQID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12), CEGRRARVC(SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ ID NO:15),CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC (SEQ IDNO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC(SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ ID NO:23),CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), or CSEWPQNVC (SEQ IDNO:26). In some embodiments, the peptide is a circular peptide. In someembodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines).

In some embodiments, the polynucleotide encodes a peptide comprising anamino acid sequence comprising any one of SEQ ID NO:27-61. In someembodiments, polynucleotide encodes a peptide comprising an amino acidsequence consisting of any one of SEQ ID NO:27-61 (including no furtheramino acid residues). In some embodiments, polynucleotide encodes apeptide consisting of an amino acid sequence consisting of any one ofSEQ ID NO:27-61. In some embodiments, the peptide is a circular peptide.In some embodiments, the peptide has an N-terminal and C-terminaldisulfide bridge (i.e., a disulfide bridge between the cysteines).

As used herein, the term “expression construct” refers to a combinationof nucleic acid sequences that provides for transcription of an operablylinked nucleic acid sequence. As used herein, the term “operably linked”refers to a juxtaposition of the components described wherein thecomponents are in a relationship that permits them to function in theirintended manner. In general, operably linked components are incontiguous relation.

Expression constructs of the invention will also generally includeregulatory elements that are functional in the intended host cell inwhich the expression construct is to be expressed. Thus, a person ofordinary skill in the art can select regulatory elements for use in, forexample, bacterial host cells, yeast host cells, plant host cells,insect host cells, mammalian host cells, and human host cells.Regulatory elements include promoters, transcription terminationsequences, translation termination sequences, enhancers, andpolyadenylation elements.

An expression construct of the invention can comprise a promotersequence operably linked to a polynucleotide sequence encoding a peptideof the invention. Promoters can be incorporated into a polynucleotideusing standard techniques known in the art. Multiple copies of promotersor multiple promoters can be used in an expression construct of theinvention. In a preferred embodiment, a promoter can be positioned aboutthe same distance from the transcription start site as it is from thetranscription start site in its natural genetic environment. Somevariation in this distance is permitted without substantial decrease inpromoter activity. A transcription start site is typically included inthe expression construct.

For expression in human or animal cells, an expression construct of theinvention can comprise suitable promoters that can drive transcriptionof the polynucleotide sequence. If the cells are mammalian cells, thenpromoters such as, for example, actin promoter, metallothioneinpromoter, NF-kappaB promoter, EGR promoter, SRE promoter, IL-2 promoter,NFAT promoter, osteocalcin promoter, SV40 early promoter and SV40 latepromoter, Lck promoter, BMP5 promoter, TRP-1 promoter, murine mammarytumor virus long terminal repeat promoter, STAT promoter, or animmunoglobulin promoter can be used in the expression construct. Thebaculovirus polyhedrin promoter can be used with an expression constructof the invention for expression in insect cells. Promoters suitable foruse with an expression construct of the invention in yeast cellsinclude, but are not limited to, 3-phosphoglycerate kinase promoter,glyceraldehyde-3-phosphate dehydrogenase promoter, metallothioneinpromoter, alcohol dehydrogenase-2 promoter, and hexokinase promoter.

For expression in prokaryotic systems, an expression construct of theinvention can comprise promoters such as, for example, alkalinephosphatase promoter, tryptophan (trp) promoter, lambda P_(L) promoter,β-lactamase promoter, lactose promoter, phoA promoter, T3 promoter, T7promoter, or tac promoter (de Boer et al., 1983).

If the expression construct is to be provided in a plant cell, plantviral promoters, such as, for example, the cauliflower mosaic virus(CaMV) 35S (including the enhanced CaMV 35S promoter (see, for exampleU.S. Pat. No. 5,106,739)) or 19S promoter can be used. Plant promoterssuch as prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA1′- or 2′-promoter of A. tumefaciens, polygalacturonase promoter,chalcone synthase A (CHS-A) promoter from petunia, tobacco PR-1apromoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2promoter (Xu et al., 1993), maize WipI promoter, maize trpA genepromoter (U.S. Pat. No. 5,625,136), maize CDPK gene promoter, andRUBISCO SSU promoter (U.S. Pat. No. 5,034,322) can also be used.Seed-specific promoters such as the promoter from a β-phaseolin gene (ofkidney bean) or a glycinin gene (of soybean), and others, can also beused. Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOSpromoter), tissue-specific promoters (such as the E8 promoter fromtomato), developmentally-regulated promoters, and inducible promoters(such as those promoters than can be induced by heat, light, hormones,or chemicals) are contemplated for use with the polynucleotides of theinvention.

Expression constructs of the invention may optionally contain atranscription termination sequence, a translation termination sequence,signal peptide sequence, and/or enhancer elements. Transcriptiontermination regions can typically be obtained from the 3′ untranslatedregion of a eukaryotic or viral gene sequence. Transcription terminationsequences can be positioned downstream of a coding sequence to providefor efficient termination. Signal peptides are a group of short aminoterminal sequences that encode information responsible for therelocation of an operably linked peptide to a wide range ofpost-translational cellular destinations, ranging from a specificorganelle compartment to sites of protein action and the extracellularenvironment. Targeting a peptide to an intended cellular and/orextracellular destination through the use of operably linked signalpeptide sequence is contemplated for use with the peptides of theinvention. Chemical enhancers are cis-acting elements that increase genetranscription and can also be included in the expression construct.Chemical enhancer elements are known in the art, and include, but arenot limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV)early promoter enhancer element, and the SV40 enhancer element. DNAsequences which direct polyadenylation of the mRNA encoded by thestructural gene can also be included in the expression construct.

Unique restriction enzyme sites can be included at the 5′ and 3′ ends ofthe expression construct to allow for insertion into a polynucleotidevector. As used herein, the term “vector” refers to any genetic element,including for example, plasmids, cosmids, chromosomes, phage, virus, andthe like, which is capable of replication when associated with propercontrol elements and which can transfer polynucleotide sequences betweencells. Vectors contain a nucleotide sequence that permits the vector toreplicate in a selected host cell. A number of vectors are available forexpression and/or cloning, and include, but are not limited to, pBR322,pUC series, M13 series, and pBLUESCRIPT vectors (Stratagene, La Jolla,Calif.).

Polynucleotides, viral and non-viral vectors, and other expressionconstructs of the subject invention can be introduced into a cell bymethods known in the art. Such methods include transfection,microinjection, electroporation, lipofection, cell fusion, calciumphosphate precipitation, and by biolistic methods. In one embodiment, apolynucleotide or expression construct of the invention can beintroduced in vivo via a viral vector such as adeno-associated virus(AAV), herpes simplex virus (HSV), papillomavirus, adenovirus, andEpstein-Barr virus (EBV). Attenuated or defective forms of viral vectorsthat can be used with the subject invention are known in the art.Typically, defective virus is not capable of infection after the virusis introduced into a cell. Polynucleotides, vectors, and expressionconstructs of the invention can also be introduced in vivo vialipofection (DNA transfection via liposomes prepared from syntheticcationic lipids) (Feigner et al., 1987, Proc Natl Acad Sci U.S.A.84(21):7413-7417). Synthetic cationic lipids (LIPOFECTIN, InvitrogenCorp., La Jolla, Calif.) can be used to prepare liposomes to encapsulatea polynucleotide, vector, or expression construct of the invention. Apolynucleotide, vector, or expression construct of the invention canalso be introduced in vivo as naked DNA using methods known in the art,such as transfection, microinjection, electroporation, calcium phosphateprecipitation, and by biolistic methods.

Polynucleotides and peptides of the subject invention can also bedefined in terms of more particular identity and/or similarity rangeswith those exemplified herein. The sequence identity will typically begreater than 60%, preferably greater than 75%, more preferably greaterthan 80%, even more preferably greater than 90%, and can be greater than95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequenceexemplified herein. Unless otherwise specified, as used herein percentsequence identity and/or similarity of two sequences can be determinedusing the algorithm of Karlin and Altschul (1990), modified as in Karlinand Altschul (1993). Such an algorithm is incorporated into the NBLASTand)(BLAST programs of Altschul et al. (1990). BLAST searches can beperformed with the NBLAST program, score=100, wordlength=12, to obtainsequences with the desired percent sequence identity. To obtain gappedalignments for comparison purposes, Gapped BLAST can be used asdescribed in Altschul et al. (1997). When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs(NBLAST and)(BLAST) can be used. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide molecules(encoding peptides of the invention) having sequences which aresufficiently homologous with the polynucleotide sequences encoding apeptide of the invention so as to permit hybridization with thatsequence under standard stringent conditions and standard methods(Maniatis, T. et al., 1982). As used herein, “stringent” conditions forhybridization refers to conditions wherein hybridization is typicallycarried out overnight at 20-25 C below the melting temperature (Tm) ofthe DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/mldenatured DNA. The melting temperature is described by the followingformula (Beltz, G. A. et al., 1983):Tm=81.5C+16.6 Log[Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm-20 C for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

As used herein, the terms “nucleic acid”, “polynucleotide”, and“polynucleotide sequence” refer to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, would encompass known analogs of naturalnucleotides that can function in a similar manner as naturally-occurringnucleotides. The polynucleotide sequences include both the DNA strandsequence that is transcribed into RNA and the RNA sequence that istranslated into protein. The polynucleotide sequences include bothfull-length sequences as well as shorter sequences derived from thefull-length sequences. It is understood that a particular polynucleotidesequence includes the degenerate codons of the native sequence orsequences which may be introduced to provide codon preference in aspecific host cell. The polynucleotide sequences falling within thescope of the subject invention further include sequences whichspecifically hybridize with the sequences coding for a peptide of theinvention. The polynucleotide includes both the sense and antisensestrands as either individual strands or in the duplex.

The subject invention also concerns a method for modulating BK channelactivity in a cell having a BK channel in vitro or in vivo. In someembodiments, the BK channel activity is BK alpha subunit (BKα) channelfunction.

In one embodiment, a cell is contacted with an effective amount of oneor more peptide, polypeptide construct, polynucleotide, or a compositionof the invention. In one embodiment, the peptide has the amino acidsequence of CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC(SEQ ID NO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQ ID NO:5),CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQ IDNO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC (SEQID NO:11), CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13). CLDGKLDVC(SEQ ID NO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ ID NO:16),CVWVKRNVC (SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC (SEQ IDNO:19), CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC (SEQ ID NO:21), CTQAETRVC(SEQ ID NO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ ID NO:24),CQAREVLVC (SEQ ID NO:25), CSEWPQNVC (SEQ ID NO:26), or a fragment orvariant thereof that exhibits BK channel modulatory activity (e.g.,modulation of BKα channel function). In some embodiments, the peptidehas an N-terminal and C-terminal disulfide bridge (i.e., a disulfidebridge between the cysteines). In some embodiments, the peptidecomprises an amino acid sequence consisting of CARGVYRVC (SEQ ID NO:1),CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ IDNO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC(SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12),CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ IDNO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC(SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20),CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ IDNO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), or CSEWPQNVC(SEQ ID NO:26).

In some embodiments, the peptide comprises an amino acid sequencecomprising any one of SEQ ID NO:27-61. In some embodiments, the peptidecomprises an amino acid sequence consisting of any one of SEQ IDNO:27-61 (including no further amino acid residues). In someembodiments, the peptide consists of an amino acid sequence consistingof any one of SEQ ID NO:27-61.

In some embodiments, the peptide is a circular peptide. In someembodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines).

The cell can be a human or mammalian cell. In some embodiments, the cellis a neuron or inner ear cell. In some embodiments, the cell is the cellof an inner ear organ, which includes the inner ear hair cell and theouter ear hair cell. In some embodiments, the cell is a spiral ganglionneuron.

Peptides, polynucleotides, compositions, and/or other agents of theinvention can be delivered to a cell either through direct contact ofpeptide, etc. with the cell or via a carrier means. Carrier means fordelivering compositions to cells are known in the art and includeencapsulating the composition in a liposome moiety, and attaching thepeptide or polynucleotide to a protein or nucleic acid that is targetedfor delivery to the target cell. Published U.S. Patent Application Nos.20030032594 and 20020120100 disclose amino acid sequences that can becoupled to another peptide, protein, or nucleic acid and that allows thepeptide, protein, or nucleic acid to be translocated across biologicalmembranes. Published U.S. Patent Application No. 20020035243 alsodescribes compositions for transporting biological moieties, such aspeptides and proteins across cell membranes for intracellular delivery.Peptides can also be delivered using a polynucleotide that encodes asubject peptide. In one embodiment, the polynucleotide is delivered tothe cell where it is taken up and the polynucleotide is transcribed intoRNA and the RNA is translated into the encoded peptide. Methods of theinvention can be conducted in vitro or in vivo.

Another aspect of the invention concerns a method for treating acondition in a subject in need thereof, comprising administering anagent of the invention to the subject. In one embodiment, the conditionis age-related hearing loss (presbycusis). In a further embodiment, thecondition is audiogenic seizures. In a further embodiment, the conditionis alcohol addiction. In a further embodiment, the condition is cancer.In a further embodiment, the condition is neurodegenerative disease(e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington'sdisease). In one embodiment, an effective amount of the agent isadministered to a subject having the condition and who is in need oftreatment thereof. In another embodiment, the subject is a person ornon-human animal at risk of developing the condition.

The agent may be a peptide of the invention, a nucleic acid encoding thepeptide, or an expression construct comprising a nucleic acid encodingthe peptide. The agent may be administered to the subject in acomposition comprising the agent and a pharmaceutically acceptablecarrier or diluent.

In one embodiment, the peptide has the amino acid sequence of CARGVYRVC(SEQ ID NO:1), CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3),CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ IDNO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQID NO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC(SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14),CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ IDNO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC(SEQ ID NO:20), CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22),CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ IDNO:25), CSEWPQNVC (SEQ ID NO:26), or a fragment or variant thereof thatexhibits BK channel modulatory activity (e.g., modulation of BKα channelfunction). In some embodiments, the peptide has an N-terminal andC-terminal disulfide bridge (i.e., a disulfide bridge between thecysteines). In some embodiments, the peptide comprises an amino acidsequence consisting of CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ ID NO:2),CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQ IDNO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC(SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13).CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ IDNO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC(SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC (SEQ ID NO:21),CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ IDNO:24), CQAREVLVC (SEQ ID NO:25), or CSEWPQNVC (SEQ ID NO:26). In someembodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines).

Methods of the invention can also further comprise administering one ormore biological molecules, compounds or drugs useful for treating thecondition. Such biological molecules, compounds or drugs can beadministered prior to, in conjunction with, and/or subsequent toadministration of a peptide, polynucleotide, and/or composition of thepresent invention. The subject can be a human or other mammal, such as adog, cat, or horse, or other animals having the condition. Methods ofthe invention can optionally comprise identifying that a person oranimal has or may develop a condition and is in need of treatment orprevention. Methods for administering and formulating peptides andpolynucleotides for administration to a subject are known in the art,examples of which are described herein. Peptides, polynucleotides,and/or compositions of the invention can be delivered to a cell eitherthrough direct contact of peptide, polynucleotide, or composition withthe cell or via a carrier means. In one embodiment, a peptide,polynucleotide, or composition of the invention comprises an attachedgroup that enhances cellular uptake of the peptide. In one embodiment,the peptide, polynucleotide, or composition is attached to an antibodythat binds to a targeted cell. In another embodiment, the peptide,polynucleotide, or composition is encapsulated in a liposome. Peptidescan also be delivered using a polynucleotide that encodes a subjectpeptide. Any polynucleotide having a nucleotide sequence that encodes apeptide of the invention is contemplated within the scope of theinvention. In one embodiment, the polynucleotide is delivered to thecell where it is taken up and the polynucleotide is transcribed into RNAand the RNA is translated into the encoded peptide.

The subject invention also concerns methods for treating an oncologicaldisorder (e.g., cancer) in a subject. In one embodiment, an effectiveamount of one or more peptide, polynucleotide, or composition of thepresent invention is administered to a subject having an oncologicaldisorder and who is in need of treatment thereof. The subject inventionalso concerns methods for inhibiting the growth of a cancer cell bycontacting the cell in vitro or in vivo with an effective amount of apeptide, polynucleotide, or composition of the present invention.

In one embodiment, the peptide has an amino acid sequence specificallydisclosed herein, such as in Table 1 (SEQ ID NO:1-26), SEQ ID NO:27, ora fragment or variant of such amino acid sequences that exhibits BKchannel modulating activity. In some embodiments, the peptide has anN-terminal and C-terminal disulfide bridge (i.e., a disulfide bridgebetween the cysteines).

In some embodiments, the peptide comprises an amino acid sequencecomprising or consisting of: CXRXXYRVC (SEQ ID NO:41), CXRGXXXVC (SEQ IDNO:42), CRRXXXXVC (SEQ ID NO:43), CRXXXXAVC (SEQ ID NO:44), CXXGRXAVC(SEQ ID NO:45), CXXGXLXVC (SEQ ID NO:46), CGMXXXXVC (SEQ ID NO:47),CXXXKRXVC (SEQ ID NO:48), CXQXXXGVC (SEQ ID NO:49), CDXMXXXVC (SEQ IDNO:50), CEQRXXXVC (SEQ ID NO:51), CXEQRXXVC (SEQ ID NO:52), CXXEQRXVC(SEQ ID NO:53), CXXXEQRVC (SEQ ID NO:54), CGRRXXXVC (SEQ ID NO:55),CXGRRXXVC (SEQ ID NO:56), CXXGRRXVC (SEQ ID NO:57), CXXXGRRVC (SEQ IDNO:58), CGGGGXXVC (SEQ ID NO:59), CXGGGGXVC (SEQ ID NO:60), or CXXGGGGVC(SEQ ID NO:61), wherein X is any natural or non-natural amino acid. Insome embodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines).

Methods of the invention can also further comprise administering orcontacting a cell with one or more compounds or biological molecules fortreating an oncological disorder. Such compounds can be administeredprior to, in conjunction with, and/or subsequent to administration of apeptide, polynucleotide, and/or composition of the present invention.Methods of the invention can optionally include identifying a subjectwho is or may be in need of treatment of an oncological disorder. Thesubject can be a human or other mammal, such as a primate (monkey,chimpanzee, ape, etc.), dog, cat, cow, pig, or horse, or other animalshaving an oncological disorder. Means for administering and formulatingpeptides, polynucleotides, or compositions of the invention foradministration to a patient are known in the art, examples of which aredescribed herein.

Oncological disorders within the scope of the invention include, but arenot limited to, cancer and/or tumors of the anus, bile duct, bladder,bone, bone marrow, bowel (including colon and rectum), breast, eye, gallbladder, kidney, mouth, larynx, esophagus, stomach, testis, cervix,head, neck, ovary, lung, mesothelioma, neuroendocrine, penis, skin,spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, pancreas,prostate, blood cells (including lymphocytes and other immune systemcells), and brain. Specific cancers contemplated for treatment with thepresent invention include carcinomas, Kaposi's sarcoma, melanoma,mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer,leukemia (hairy cell, acute lymphoblastic, acute myeloid, chroniclymphocytic, chronic myeloid, and other), and lymphoma (Hodgkin's andnon-Hodgkin's), and follicular lymphoma, and multiple myeloma.

Examples of cancers that can be treated according to the presentinvention are listed in Table 4.

TABLE 4 Examples of Cancer Types Acute Lymphoblastic Leukemia, AdultHairy Cell Leukemia Acute Lymphoblastic Leukemia, Head and Neck CancerChildhood Hepatocellular (Liver) Cancer, Adult (Primary) Acute MyeloidLeukemia, Adult Hepatocellular (Liver) Cancer, Childhood Acute MyeloidLeukemia, Childhood (Primary) Adrenocortical Carcinoma Hodgkin'sLymphoma, Adult Adrenocortical Carcinoma, Childhood Hodgkin's Lymphoma,Childhood AIDS-Related Cancers Hodgkin's Lymphoma During PregnancyAIDS-Related Lymphoma Hypopharyngeal Cancer Anal Cancer Hypothalamic andVisual Pathway Glioma, Astrocytoma, Childhood Cerebellar ChildhoodAstrocytoma, Childhood Cerebral Intraocular Melanoma Basal CellCarcinoma Islet Cell Carcinoma (Endocrine Pancreas) Bile Duct Cancer,Extrahepatic Kaposi's Sarcoma Bladder Cancer Kidney (Renal Cell) CancerBladder Cancer, Childhood Kidney Cancer, Childhood Bone Cancer,Osteosarcoma/Malignant Laryngeal Cancer Fibrous Histiocytoma LaryngealCancer, Childhood Brain Stem Glioma, Childhood Leukemia, AcuteLymphoblastic, Adult Brain Tumor, Adult Leukemia, Acute Lymphoblastic,Childhood Brain Tumor, Brain Stem Glioma, Leukemia, Acute Myeloid, AdultChildhood Leukemia, Acute Myeloid, Childhood Brain Tumor, CerebellarAstrocytoma, Leukemia, Chronic Lymphocytic Childhood Leukemia, ChronicMyelogenous Brain Tumor, Cerebral Leukemia, Hairy CellAstrocytoma/Malignant Glioma, Lip and Oral Cavity Cancer Childhood LiverCancer, Adult (Primary) Brain Tumor, Ependymoma, Childhood Liver Cancer,Childhood (Primary) Brain Tumor, Medulloblastoma, Lung Cancer, Non-SmallCell Childhood Lung Cancer, Small Cell Brain Tumor, SupratentorialPrimitive Lymphoma, AIDS-Related Neuroectodermal Tumors, ChildhoodLymphoma, Burkitt's Brain Tumor, Visual Pathway and Lymphoma, CutaneousT-Cell, see Mycosis Hypothalamic Glioma, Childhood Fungoides and SézarySyndrome Brain Tumor, Childhood Lymphoma, Hodgkin's, Adult Breast CancerLymphoma, Hodgkin's, Childhood Breast Cancer, Childhood Lymphoma,Hodgkin's During Pregnancy Breast Cancer, Male Lymphoma, Non-Hodgkin's,Adult Bronchial Adenomas/Carcinoids, Lymphoma, Non-Hodgkin's, ChildhoodChildhood Lymphoma, Non-Hodgkin's During Pregnancy Burkitt's LymphomaLymphoma, Primary Central Nervous System Carcinoid Tumor, ChildhoodMacroglobulinemia, Waldenstrom's Carcinoid Tumor, GastrointestinalMalignant Fibrous Histiocytoma of Carcinoma of Unknown PrimaryBone/Osteosarcoma Central Nervous System Lymphoma, Medulloblastoma,Childhood Primary Melanoma Cerebellar Astrocytoma, Childhood Melanoma,Intraocular (Eye) Cerebral Astrocytoma/Malignant Glioma, Merkel CellCarcinoma Childhood Mesothelioma, Adult Malignant Cervical CancerMesothelioma, Childhood Childhood Cancers Metastatic Squamous NeckCancer with Occult Chronic Lymphocytic Leukemia Primary ChronicMyelogenous Leukemia Multiple Endocrine Neoplasia Syndrome, ChronicMyeloproliferative Disorders Childhood Colon Cancer MultipleMyeloma/Plasma Cell Neoplasm Colorectal Cancer, Childhood MycosisFungoides Cutaneous T-Cell Lymphoma, see Myelodysplastic SyndromesMycosis Fungoides and Sézary Myelodysplastic/Myeloproliferative DiseasesSyndrome Myelogenous Leukemia, Chronic Endometrial Cancer MyeloidLeukemia, Adult Acute Ependymoma, Childhood Myeloid Leukemia, ChildhoodAcute Esophageal Cancer Myeloma, Multiple Esophageal Cancer, ChildhoodMyeloproliferative Disorders, Chronic Ewing's Family of Tumors NasalCavity and Paranasal Sinus Cancer Extracranial Germ Cell Tumor,Nasopharyngeal Cancer Childhood Nasopharyngeal Cancer, ChildhoodExtragonadal Germ Cell Tumor Neuroblastoma Extrahepatic Bile Duct CancerNon-Hodgkin's Lymphoma, Adult Eye Cancer, Intraocular MelanomaNon-Hodgkin's Lymphoma, Childhood Eye Cancer, RetinoblastomaNon-Hodgkin's Lymphoma During Pregnancy Gallbladder Cancer Non-SmallCell Lung Cancer Gastric (Stomach) Cancer Oral Cancer, Childhood Gastric(Stomach) Cancer, Childhood Oral Cavity Cancer, Lip and GastrointestinalCarcinoid Tumor Oropharyngeal Cancer Germ Cell Tumor, Extracranial,Osteosarcoma/Malignant Fibrous Histiocytoma Childhood of Bone Germ CellTumor, Extragonadal Ovarian Cancer, Childhood Germ Cell Tumor, OvarianOvarian Epithelial Cancer Gestational Trophoblastic Tumor Ovarian GermCell Tumor Glioma, Adult Ovarian Low Malignant Potential Tumor Glioma,Childhood Brain Stem Pancreatic Cancer Glioma, Childhood CerebralPancreatic Cancer, Childhood Astrocytoma Pancreatic Cancer, Islet CellGlioma, Childhood Visual Pathway and Paranasal Sinus and Nasal CavityCancer Hypothalamic Parathyroid Cancer Skin Cancer (Melanoma) PenileCancer Skin Carcinoma, Merkel Cell Pheochromocytoma Small Cell LungCancer Pineoblastoma and Supratentorial Primitive Small Intestine CancerNeuroectodermal Tumors, Childhood Soft Tissue Sarcoma, Adult PituitaryTumor Soft Tissue Sarcoma, Childhood Plasma Cell Neoplasm/MultipleMyeloma Squamous Cell Carcinoma, see Skin Pleuropulmonary BlastomaCancer (non-Melanoma) Pregnancy and Breast Cancer Squamous Neck Cancerwith Occult Pregnancy and Hodgkin's Lymphoma Primary, MetastaticPregnancy and Non-Hodgkin's Lymphoma Stomach (Gastric) Cancer PrimaryCentral Nervous System Lymphoma Stomach (Gastric) Cancer, ChildhoodProstate Cancer Supratentorial Primitive Rectal Cancer NeuroectodermalTumors, Childhood Renal Cell (Kidney) Cancer T-Cell Lymphoma, Cutaneous,see Renal Cell (Kidney) Cancer, Childhood Mycosis Fungoides and SézaryRenal Pelvis and Ureter, Transitional Cell Syndrome Cancer TesticularCancer Retinoblastoma Thymoma, Childhood Rhabdomyosarcoma, ChildhoodThymoma and Thymic Carcinoma Salivary Gland Cancer Thyroid CancerSalivary Gland Cancer, Childhood Thyroid Cancer, Childhood Sarcoma,Ewing's Family of Tumors Transitional Cell Cancer of the Renal Sarcoma,Kaposi's Pelvis and Ureter Sarcoma, Soft Tissue, Adult TrophoblasticTumor, Gestational Sarcoma, Soft Tissue, Childhood Unknown Primary Site,Carcinoma of, Sarcoma, Uterine Adult Sezary Syndrome Unknown PrimarySite, Cancer of, Skin Cancer (non-Melanoma) Childhood Skin Cancer,Childhood Unusual Cancers of Childhood Ureter and Renal Pelvis,Transitional Cell Cancer Urethral Cancer Uterine Cancer, EndometrialUterine Sarcoma Vaginal Cancer Visual Pathway and Hypothalamic Glioma,Childhood Vulvar Cancer Waldenström's Macroglobulinemia Wilms' Tumor

For the treatment of oncological disorders (cancers), the peptides,polynucleotides, and compositions of this invention can be administeredto a patient in need of treatment in combination with other antitumor oranticancer substances and/or with radiation and/or photodynamic therapyand/or with surgical treatment to remove a tumor. These other substancesor treatments may be given at the same as or at different times from thepeptides, polynucleotides, and compositions of this invention. Forexample, the peptides, polynucleotides, and compositions of the presentinvention can be used in combination with mitotic inhibitors such astaxol or vinblastine, alkylating agents such as cyclophosamide orifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNAintercalators such as adriamycin or bleomycin, topoisomerase inhibitorssuch as etoposide or camptothecin, antiangiogenic agents such asangiostatin, antiestrogens such as tamoxifen, and/or other anticancerdrugs or antibodies, such as, for example, GLEEVEC (NovartisPharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.),respectively. Peptides, polynucleotides, and compositions of theinvention can be used in combination with proteasome inhibitors,including, but not limited to, Bortezomib, Carfilzomib, andSalinosporamide A. The subject invention also concerns methods forinhibiting the growth of a cancer cell by contacting the cell in vitroor in vivo with an effective amount of a peptide, polynucleotide, orcomposition of the present invention.

The methods of the present invention can be used with humans andnon-human animals. The animals contemplated within the scope of theinvention include domesticated, agricultural, or zoo- orcircus-maintained animals. Domesticated animals include, for example,dogs, cats, rabbits, ferrets, guinea pigs, hamsters, pigs, monkeys orother primates, and gerbils. Agricultural animals include, for example,horses, mules, donkeys, burros, cattle, cows, pigs, sheep, andalligators. Zoo- or circus-maintained animals include, for example,lions, tigers, bears, camels, giraffes, hippopotamuses, andrhinoceroses.

In one embodiment, one or more of the peptides of the subject inventioncan be provided in the form of a multiple peptide construct. Such aconstruct can be designed so that multiple peptides are linked to eachother by intervening moieties wherein the intervening moieties aresubsequently cleaved or removed following administration of the multiplepeptide construct to a subject. Methods for constructing multiplepeptide constructs are known in the art. For example, peptides of thepresent invention can be provided in the form resembling a multipleantigenic peptide (MAP) construct. The preparation of MAP constructs hasbeen described in Tam J P, 1988, Biochemistry 85:5409-5413. MAPconstructs utilize a core matrix of lysine residues onto which multiplecopies of an immunogen are synthesized. Multiple peptide constructs,each containing different peptides, can be prepared and administered inaccordance with methods of the present invention. In another embodiment,a multiple peptide construct can be prepared by preparing the subjectpeptides having at least one metal chelating amino acid incorporatedtherein, preferably at the amino and/or carboxy terminal of the peptideas described, for example, in U.S. Pat. No. 5,763,585. The peptides arethen contacted with a solid support having attached thereto a metal ionspecific for the metal chelating amino acid of the peptide. A multiplepeptide construct of the invention can provide multiple copies of theexact same peptide, including variants or fragments of a subjectpeptide, or copies of different peptides of the subject invention.

Therapeutic application of the subject peptides, polynucleotides, andcompositions containing them, can be accomplished by any suitabletherapeutic method and technique presently or prospectively known tothose skilled in the art. The peptides, polynucleotides, andcompositions can be administered by any suitable route known in the artincluding, for example, topical, oral, nasal, rectal, parenteral,subcutaneous, or intravascular (e.g., intravenous or intra-arterial)routes of administration. For example, the peptides, polynucleotides andcompositions can be administered to the subject systemically or locally.For presbycusis, for example, the peptides, polynucleotides, andcompositions can be administered systemically (e.g., intravascularly) orlocally, such as at the site of the inner ear (inner ear cell, outer earhair cell, supporting cell, spiral ganglion cell, etc.). Administrationof the peptides, polynucleotides, and compositions of the invention canbe continuous or at distinct intervals as can be readily determined by aperson skilled in the art.

Agents and compositions useful in the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W. Martindescribes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of the bioactive peptide orpolynucleotide is combined with a suitable carrier in order tofacilitate effective administration of the composition. The compositionsused in the present methods can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the subject peptides and polynucleotides include, but are notlimited to, water, saline, oils including mineral oil, ethanol, dimethylsulfoxide, gelatin, cyclodextrans, magnesium stearate, dextrose,cellulose, sugars, calcium carbonate, glycerol, alumina, starch, andequivalent carriers and diluents, or mixtures of any of these.Formulations of the peptide or polynucleotide of the invention can alsocomprise suspension agents, protectants, lubricants, buffers,preservatives, and stabilizers. To provide for the administration ofsuch dosages for the desired therapeutic treatment, pharmaceuticalcompositions of the invention will advantageously comprise between about0.1% and 45%, and especially, 1 and 15% by weight of the total of one ormore of the peptide or polynucleotide based on the weight of the totalcomposition including carrier or diluent.

The peptides, polynucleotides, and compositions of the subject inventioncan also be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time.

The subject peptides and polynucleotides can also be modified by theaddition of chemical groups, such as PEG (polyethylene glycol).PEGylated peptides typically generate less of an immunogenic responseand exhibit extended half-lives in vivo in comparison to peptides thatare not PEGylated when administered in vivo. Methods for PEGylatingproteins and peptides known in the art (see, for example, U.S. Pat. No.4,179,337). The subject peptides and polynucleotides can also bemodified to improve cell membrane permeability. In one embodiment, cellmembrane permeability can be improved by attaching a lipophilic moiety,such as a steroid, to the peptide or polynucleotide. In anotherembodiment, peptides and polynucleotides of the invention comprise acell-penetrating peptide (CPP). CPPs are typically short peptides thatare highly cationic and typically include several arginine and/or lysineamino acids. CPPs can be classified as hydrophilic, amphiphilic, orperiodic sequence. In one embodiment, a CPP is provided at the terminusof a peptide or polynucleotide.

The subject invention also concerns a packaged dosage formulationcomprising in one or more containers at least one peptide,polynucleotide, and/or composition of the subject invention formulatedin a pharmaceutically acceptable dosage. The package can containdiscrete quantities of the dosage formulation, such as tablet, capsules,lozenge, and powders. The quantity of peptide and/or polynucleotide in adosage formulation and that can be administered to a patient can varyfrom about 1 mg to about 5000 mg, or about 1 mg to about 2000 mg, ormore typically about 1 mg to about 500 mg, or about 5 mg to about 250mg, or about 10 mg to about 100 mg.

The subject invention also concerns kits comprising one or morepeptides, polynucleotides, compositions, compounds, or molecules of thepresent invention in one or more containers. In one embodiment, a kitcontains a peptide, polynucleotide, and/or composition of the presentinvention. In a specific embodiment, a kit comprises a peptidecomprising the amino acid sequence CARGVYRVC (SEQ ID NO:1), CRVAHRAVC(SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ ID NO:4),CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQ IDNO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC (SEQID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12), CEGRRARVC(SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ ID NO:15),CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC (SEQ IDNO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC(SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ ID NO:23),CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), CSEWPQNVC (SEQ IDNO:26), or a fragment or variant of the peptide that exhibitssubstantially the same activity as the full-length non-variant peptide,such as BK channel modulatory activity (e.g., modulation of BKα channelfunction). In some embodiments, the peptide is a circular peptide. Insome embodiments, the peptide has an N-terminal and C-terminal disulfidebridge (i.e., a disulfide bridge between the cysteines).

A kit of the invention can also comprise, in addition to a peptide,polynucleotide, and/or composition of the invention, one or morecompounds, biological molecules, or drugs for treating presbycusis,audiogenic seizures, alcohol addiction, cancer, or neurodegenerativedisease.

In one embodiment, a kit of the invention includes instructions orpackaging materials that describe how to administer a peptide,polynucleotide, compositions, compounds, or molecules of the kit.Containers of the kit can be of any suitable material, e.g., glass,plastic, metal, etc., and of any suitable size, shape, or configuration.In one embodiment, a peptide, polynucleotide, compositions, compounds,or molecules of the invention is provided in the kit as a solid, such asa tablet, pill, or powder form. In another embodiment, a peptide,polynucleotide, compositions, compounds, or molecules of the inventionis provided in the kit as a liquid or solution. In one embodiment, thekit comprises an ampoule or syringe containing a peptide,polynucleotide, compositions, compounds, or molecules of the inventionin liquid or solution form.

The subject invention can be used in gene therapy to treat a condition(presbycusis, audiogenic seizures, alcohol addiction, cancer, orneurodegenerative disease) in a person or animal subject. In oneembodiment, a polynucleotide of the invention is incorporated into acell or cells of a person or animal subject, and the polynucleotideexpressed in the cell to produce a peptide of the invention. In aspecific embodiment, a cell is removed from the body of the person oranimal, the polynucleotide is incorporated into the cell ex vivo, andthe cell is then reintroduced back into the body of the person or animaland the polynucleotide expressed in the cell. In one embodiment, thepolynucleotide is stably incorporated into the genome of the cell. In aspecific embodiment, the polynucleotide is provided in an expressionconstruct that provides for expression of the polynucleotide in thecell. In one embodiment, the peptide expressed in the cell istransported outside the cell and into the extracellular space of theperson or animal.

Any methods of the subject invention can optionally include a step ofidentifying a person or animal who is or who may be in need of treatmentor prevention of a condition prior to administration of the peptide,polynucleotide, or composition of the invention.

As used herein, the term “peptide of the invention” includes peptidescomprising an amino acid sequence comprising any one of CARGVYRVC (SEQID NO:1), CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC(SEQ ID NO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQ ID NO:6),CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC (SEQ IDNO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC(SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ ID NO:14),CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC (SEQ IDNO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC(SEQ ID NO:20), CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ ID NO:22),CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC (SEQ IDNO:25), CSEWPQNVC (SEQ ID NO:26), or a functional fragment or variant ofany of the foregoing. In some embodiments, the peptide of the inventioncomprises an amino acid sequence consisting of any one of SEQ ID NO:1-26(including no further amino acid residues). In some embodiments, thepeptide of the invention consists of an amino acid sequence consistingof any one of SEQ ID NO:1-26. In some embodiments, the peptide of theinvention comprises an amino acid sequence comprising any one of SEQ IDNO:27-61. In some embodiments, the peptide of the invention comprises anamino acid sequence consisting of any one of SEQ ID NO:27-61 (includingno further amino acid residues). In some embodiments, the peptide of theinvention consists of an amino acid sequence consisting of any one ofSEQ ID NO:27-61. In some embodiments, the peptide of the inventioncomprises or consists of an amino acid sequence of Table 1.

The term “a,” “an,” “the” and similar terms used in the context of thepresent invention (especially in the context of the claims) are to beconstrued to cover both the singular and plural unless otherwiseindicated herein or clearly contradicted by the context. Thus, forexample, reference “a cell” or “a peptide” should be construed to coverboth a singular cell or singular peptide and a plurality of cells and aplurality of peptides unless indicated otherwise or clearly contradictedby the context.

The term “isolated,” when used as a modifier of a peptide orpolynucleotide, means that the peptides or compositions are made by thehand of man or are separated from their naturally occurring in vivoenvironment. Generally, compositions so separated are substantially freeof one or more materials with which they normally associate with innature, for example, one or more protein, nucleic acid, lipid,carbohydrate, cell membrane. A “substantially pure” molecule can becombined with one or more other molecules. Thus, the term “substantiallypure” does not exclude combinations of compositions. Substantial puritycan be at least about 60% or more of the molecule by mass. Purity canalso be about 70% or 80% or more, and can be greater, for example, 90%or more. Purity can be determined by any appropriate method, including,for example, UV spectroscopy, chromatography (e.g., HPLC, gas phase),gel electrophoresis (e.g., silver or coomassie staining) and sequenceanalysis (for nucleic acid and peptide).

As used herein, the term “administration” is intended to include, but isnot limited to, the following delivery methods: topical, includingtopical delivery to the round window membrane of the cochlea, oral,parenteral, subcutaneous, transdermal, transbuccal, intravascular (e.g.,intravenous or intra-arterial), intramuscular, subcutaneous, intranasal,and intra-ocular administration.

As used herein, the term “hearing loss” is intended to mean anyreduction in a subject's ability to detect sound. Hearing loss isdefined as a 10 decibel (dB) standard threshold shift or greater inhearing sensitivity for two of 6 frequencies ranging from 0.5-6.0 (0.5,1, 2, 3, 4, and 6) kHz (cited in Dobie, R. A. (2005) AudiometricThreshold Shift Definitions: Simulations and Suggestions, Ear andHearing 26(1) 62-77). Hearing loss can also be only high frequency, andin this case would be defined as 5 dB hearing loss at two adjacent highfrequencies (2-6 kHz), or 10 dB at any frequency above 2 kHz. Oneexample of hearing loss is age-related (or aging-related) hearing loss,which is the gradual onset of hearing loss with increasing age.

The term “prevention” or “preventing” in the context of conditions, suchas presbycusis, audiogenic seizures, alcohol addiction, cancer, andneurodegenerative disease, encompasses preventing initial onset,preventing relapse, or delaying onset of the condition. In the contextof hearing loss, specifically, it is intended to refer to a significantdecrease is the loss of hearing sensitivity within the aforesaidfrequency range, particularly at the high frequency range 4-6 kHz.

EXEMPLIFIED EMBODIMENTS

Examples of embodiments of the invention include, but are not limitedto:

Embodiment 1

A peptide comprising an amino acid sequence selected from among:CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ ID NO:2), CRRGLVQVC (SEQ IDNO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQ ID NO:5), CWKSRWYVC (SEQID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC (SEQ ID NO:8), CRRKRHAVC(SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10), CLQEQRGVC (SEQ ID NO:11),CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ ID NO:13). CLDGKLDVC (SEQ IDNO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC (SEQ ID NO:16), CVWVKRNVC(SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18), CDTMEQRVC (SEQ ID NO:19),CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC (SEQ ID NO:21), CTQAETRVC (SEQ IDNO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC (SEQ ID NO:24), CQAREVLVC(SEQ ID NO:25), CSEWPQNVC (SEQ ID NO:26), or a functional fragment orvariant of any of the foregoing.

Embodiment 2

The peptide of embodiment 1, wherein the peptide comprises CRRGLVQVC(SEQ ID NO:3).

Embodiment 3

The peptide of embodiment 1, wherein the peptide comprises an amino acidsequence consisting of CRRGLVQVC (SEQ ID NO:3).

Embodiment 4

The peptide of embodiment 1, wherein the peptide comprises an amino acidsequence comprising the formula CXXXXXXVC (SEQ ID NO:27), wherein X isany natural or non-natural amino acid.

Embodiment 5

The peptide of embodiment 1, wherein the peptide comprises an amino acidsequence consisting of the formula CXXXXXXVC (SEQ ID NO:27), wherein Xis any natural or non-natural amino acid.

Embodiment 6

The peptide of embodiment 1, wherein the peptide comprises an amino acidsequence comprising any one of SEQ ID NO:28-61, wherein X is any naturalor non-natural amino acid.

Embodiment 7

The peptide of embodiment 1, wherein the peptide comprises an amino acidsequence consisting of any one of SEQ ID NO:28-61, wherein X is anynatural or non-natural amino acid.

Embodiment 8

The peptide of embodiment 1, wherein the peptide consists of an aminoacid sequence consisting of any one of SEQ ID NO:28-61, wherein X is anynatural or non-natural amino acid.

Embodiment 9

The peptide of embodiment 1, wherein the peptide comprises an amino acidsequence selected from among: CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ IDNO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC(SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10),CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ IDNO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC(SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18),CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC (SEQ IDNO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC(SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), and CSEWPQNVC (SEQ ID NO:26).

Embodiment 10

The peptide of embodiment 1, wherein the peptide comprises an amino acidsequence consisting of: CARGVYRVC (SEQ ID NO:1), CRVAHRAVC (SEQ IDNO:2), CRRGLVQVC (SEQ ID NO:3), CPPGRGAVC (SEQ ID NO:4), CGMTKRPVC (SEQID NO:5), CWKSRWYVC (SEQ ID NO:6), CERRMYRVC (SEQ ID NO:7), CRRAYEMVC(SEQ ID NO:8), CRRKRHAVC (SEQ ID NO:9), CAVGRLAVC (SEQ ID NO:10),CLQEQRGVC (SEQ ID NO:11), CRKQGRRVC (SEQ ID NO:12), CEGRRARVC (SEQ IDNO:13). CLDGKLDVC (SEQ ID NO:14), CGGGGSRVC (SEQ ID NO:15), CFTGGGGVC(SEQ ID NO:16), CVWVKRNVC (SEQ ID NO:17), CGMASSFVC (SEQ ID NO:18),CDTMEQRVC (SEQ ID NO:19), CGQQSPGVC (SEQ ID NO:20), CDEMNWWVC (SEQ IDNO:21), CTQAETRVC (SEQ ID NO:22), CPKPNNTVC (SEQ ID NO:23), CVRAPPSVC(SEQ ID NO:24), CQAREVLVC (SEQ ID NO:25), or CSEWPQNVC (SEQ ID NO:26).

Embodiment 11

The peptide of any preceding embodiment, wherein the peptide is acircular peptide.

Embodiment 12

The peptide of embodiment 11, wherein the peptide has a disulfide bridgebetween cysteines.

Embodiment 13

The peptide of any one of embodiments 1 to 12, further comprising aheterologous amino acid sequence, or other moiety, fused directly orindirectly to the amino acid sequence.

Embodiment 14

The peptide of embodiment 13, wherein the moiety comprises a detectablelabel.

Embodiment 15

A nucleic acid encoding a peptide of any one of embodiments 1 to 13.

Embodiment 16

An expression construct comprising the nucleic acid of embodiment 15.

Embodiment 17

A composition comprising a peptide of any one of embodiments 1 to 14,the nucleic acid of embodiment 15, or the expression construct ofembodiment 16; and a pharmaceutically acceptable carrier or diluent.

Embodiment 18

A method for treating a condition in a subject in need thereof,comprising administering an agent to the subject, wherein the agentcomprises: a peptide of any one of embodiments 1 to 13, the nucleic acidof embodiment 15, the expression construct of embodiment 16, or thecomposition of embodiment 17, and wherein the condition is selected fromamong presbycusis, audiogenic seizures, alcohol addiction, cancer, andneurodegenerative disease.

Embodiment 19

The method of embodiment 18, wherein the agent comprises a peptidecomprising CRRGLVQVC (SEQ ID NO:3).

Embodiment 20

The method of embodiment 18, wherein the agent comprises a peptidecomprising an amino acid sequence consisting of CRRGLVQVC (SEQ ID NO:3).

Embodiment 21

The method of embodiment 18 or 19, wherein the condition is presbycusis.

Embodiment 22

The method of any one of embodiments 18 to 21, wherein the subject hasthe condition at the time of said administering, and the agent isadministered to the subject as therapy.

Embodiment 23

The method of any one of embodiments 18 to 22, wherein the subject doesnot have the condition at the time of said administering, and the agentis administered to the subject as prophylaxis.

Embodiment 24

The method of any one of embodiments 18 to 23, wherein the agent isadministered to the subject by a route selected from the groupconsisting of intravascular (e.g., intravenous or intra-arterial),intramuscular, subcutaneous, oral, intranasal, intra-ocular, topical,and transdermal.

Embodiment 25

The method of any one of embodiments 18 to 24, wherein the subject is ahuman.

Embodiment 26

A method for modulating large conductance Ca²⁺ activated K⁺ (BK) channelactivity in a cell having a BK channel in vitro or in vivo, comprisingcontacting the cell in vitro or in vivo with an agent that comprises apeptide of any one of embodiments 1 to 13, the nucleic acid ofembodiment 15, the expression construct of embodiment 16, or thecomposition of embodiment 17.

Embodiment 27

The method of embodiment 26, wherein the BK channel is native to thecell.

Embodiment 28

The method of embodiment 26, wherein the BK channel is heterologous tothe cell and the cell has been genetically modified to express theheterologous BK channel.

Embodiment 29

The method of embodiment 26, wherein the cell is a human cell.

Embodiment 30

A method of delivering a cargo moiety to the brain of a subject, throughthe blood-brain barrier (BBB), comprising administering a polypeptideconstruct to the subject, wherein the polypeptide construct comprises apeptide of any one of embodiments 1 to 13 conjugated to the cargomoiety.

Embodiment 31

The method of embodiment 30, wherein the cargo moiety comprises adetectable label.

Embodiment 32

The method of embodiment 30, wherein the cargo moiety comprises anucleic acid.

Embodiment 33

A cell comprising a nucleic acid encoding a peptide of any one ofembodiments 1 to 13.

Embodiment 34

The cell of embodiment 33, wherein the nucleic acid expressed to producethe peptide.

Embodiment 35

An antibody, or antibody fragment, that selectively binds to a peptideof any one of embodiments 1 to 13.

Materials and Methods

Peptide Selection and Synthesis.

A monovalent phagemid display library (library C, Mobitec) wassequentially panned against three sets of HEK293 cells (see below). Fornegative selection, cells expressed the human glycine receptor al(hGlyRα1, X52009) and the rat small conductance calcium-activatedchannel 2 (rSK2, U69882.1). For positive selection, cells expressed thehuman BKα channel ZERO isoform (NM_002238). The progress of theselection process was monitored after each panning round by titering andsequencing the phagemid DNA. With 6 randomized and 3 fixed amino acids,this library started with 3×10⁷ unique sequences. LS3 was selected asone of the sequences expressing motifs that were enriched more than 1000fold. LS3 was synthesized as a TFA salt at 98-99% purity verified byHPLC and MS analysis (Genscript, Piscataway, N.J.). Stocks weredissolved in water at 10 mM and aliquots were lyophilized and stored at−80° C. As a secondary determination of identity and purity, in-houseLC/MS was performed on a single quadrupole Mass Spectrophotometer(Agilent 6130) interfaced with a HPLC with a diode-array (UV-vis)detector (Agilent 1200).

C. elegans Strains and Transgenics.

Worms were cultivated at 20° C. as described with OP50 bacteria⁶⁷. Wormscultured on plates contaminated with fungi or other bacteria wereexcluded from this study. The reference wild-type strain was N2 Bristol.The reference slo-1(null) strain and background for transgenic strainswas NM1968, harboring the previously characterized null allele, js379²⁹.Multi-site gateway technology (Invitrogen) was used to constructplasmids. 2501 kb of the native slo-1 promoter (Pslo-1) and thetraditional unc9-54 UTR were used in combination withslo-1a(cDNA)::mCherry for a rescue construct. To test rescue with thehuman BK channel, an hslo(ZERO isoform, cDNA)::mCherry version wasconstructed. The slo-1(+) and hslo(+) plasmids were injected at aconcentration of 20 and 10 ng/μl, respectively. The co-injectionreporter PCFJ90 (1.25 ng/μl) was used to ensure proper transformation ofthe arrays. As such, JPS345 carried vxEx345, an extrachromosomal arraycontaining[Pslo-1::slo-1::mCherry::unc-54UTR,Pmyo-2::mCherry::unc-54UTR]. JPS340carried vxEx345, an extrachromosomal array containing[Pslo-1::hslo::mCherry::unc-54UTR,Pmyo-2::mCherry::unc-54UTR].

C. elegans Behavioral Assays.

Age-matched day one adults were cleaned of bacteria by letting themcrawl around on an unseeded plate and then moved into a puddle of NGM orpeptide dissolved in NGM on another unseeded plate. NGM and peptidetreatment groups were always run in tandem to control for behavioralvariance. While much shorter than typical drug applications in C.elegans ^(68,69), brief application in liquid was chosen to avoidpotential catabolic by-products of incubation on metabolically active E.coli. Higher LS3 concentrations were used to compensate. The puddle wasrefreshed 1-2 times as needed, but let to fully absorb into the agar by30 minutes. After 30 minutes, crawl behavior was videoed (Flea2 camera,Point Grey Research, Canada; StreamPix 3, NorPix, Canada). Copper ringsrestricted movement to a proscribed area. The worms were tracked offlineusing custom macros (Image-Pro, MediaCybernetics, Rockville, Md.) for 1minute to obtain crawl speed (cm/min). Group means±SEM forpeptide-treated vs. vehicle-treated controls were compared at eachconcentration with Student's t-tests. Rescue analysis was completed withtwo-way ANOVA (SigmaPlot, San Jose, Calif.). Crawl speeds for thepeptide treated groups were also normalized to the performance of yokedcontrols. Normalized group means±SEM were compared vs. slo-1 nullperformance by two-way ANOVA.

HEK Cell Maintenance and Transfection.

HEK293 cells (ATCC, Manassas, Va.) were grown according to standardprocedures. Cells were cultured at 37° C. in a 5% CO2 atmosphere inDulbecco's modified Eagle's medium with 1-glutamine, sodium pyruvate and10% fetal bovine serum (Invitrogen). Cell lines were split withtrypsin/EDTA in Hanks' balanced salt solution (Invitrogen) up to 25-30cycles. For phage display, stable lines stably expressed rSK2 or hBKαZERO isoform. Cells were transfected (Lipofectamine 2000, Invitrogen)with hGlyRα1 and used 48 hours later. For electrophysiologicalrecordings, cells were transfected with the hBKα ZERO or STREX isoform.Enhanced green fluorescent protein (EGFP) was cotransfected as a marker.Electrophysiological recordings were made 16-72 h after transfection.Although the profile of BK channel composition varies from tissue totissue, the ZERO BK channel splice variant is widely expressed,particularly in the nervous system, serving as a representative form forstudying the modulation of BK channel gating.

Patch-Clamp Recordings.

Voltage-clamp recordings were performed at room temperature (22-24° C.)using an inside-out configuration on patches pulled from HEK293 cells.The extracellular solution contained the following (in mM): 2 KCl, 136KOH, 20 Hepes, 2 MgCl₂, adjusted to pH 7.2 with MeSO₃H. In order toapply peptide to the extracellular surface, patch electrodes (7-20 MΩ inresistance) were tip filled with normal extracellular solution andbackfilled with extracellular solution containing LS3. Enough normalextracellular solution was included to provide at least five minutes ofpeptide-free recording (determined by plotting P_(o) vs. time). Theintracellular solution (in the bath) contained the following (in mM): 6KCl, 132 KOH, 20 Hepes, adjusted to pH 7.2 with MeSO₃H. To achieve 750nM free Ca²⁺, 4.17 mL of 1 M CaCl₂ and 5 mM EGTA were included, a ratioverified by measurement with a Ca²⁺-sensitive electrode. Voltage-clamprecordings made with an Axopatch 200A amplifier and custom macros inIgorPro. Analysis was performed with QUB (www.qub.buffalo.edu),including P_(o), mean open time and three component exponential fits toclosed dwell times. Group means±SEM for post peptide measures wereplotted relative to pre peptide values and compared with pre values viaplanned paired t-tests.

Receptor Binding Assay.

LS3 was screened against a comprehensive panel of CNS-based proteins.Detailed protocols can be found within the US National Institute ofMental Health Psychoactive Drug Screening Program (NIMH PDSP) AssayProtocol Book (version II), by B. L. Roth, March 2013 (available at:pdsp.med.unc.edu/PDSP%20Protocols%20II%202013-03-28.pdf). Briefly,competition binding assays tested whether 10 microM LS3 significantlyaltered binding of known radioligands for 33 targets. Radioactivity inthe presence of the LS3 (sample) was calculated with the followingequation and expressed as a percent inhibition: % inhibition=(sampleCPM−non-specific CPM)/Total CPM−non-specific CPM)×100. Total binding wasmeasured with no competing ligand. Non-specific binding was measured inthe presence of reference compound. The % inhibition by LS3 was measured4 times for each receptor. Less than 50% inhibition was consideredinsignificant as this suggests a Ki<10 microM.

Mouse Subjects.

Multi-channel recordings were acquired from young CBA/CaJ mice. CBA/CaJmice were chosen because the loss of peripheral function is similar tohumans, making them a good model for the study of presbycusis^(70,73).Founder breeding pairs were obtained from The Jackson Laboratory (BarHarbor, Me.), bred within the facilities of the university vivarium, andhoused 3-4 per cage with litter-mates in rodent micro-isolator cages(36.9×15.6×13.2 cm), on a 12/12 hour light/dark cycle with ad lib waterand food pellets. The temperature was maintained near 25° C. Cages werechanged weekly, and the mice were monitored for signs of distressseveral times throughout the day. Only nulliparous mice were used forexperiments, while breeder mice were kept in separate cages. Allprocedures were preapproved by the University of South Florida Committeeon Animal Resources and are consistent with US Federal and NIHguidelines under IACUC protocol #0245R.

Surgical Preparation.

The mice were initially anesthetized with an intraperitoneal (i.p.)injection of ketamine and xylazine (100 mg/kg and 10 mg/kg). Afteranesthesia was induced, the top of the animal's head and neck was thenshaved of fur to prevent contamination of the incision site. The skinwas cleaned with germicidal scrub, rinsed with 70% alcohol, and preppedwith iodine. The skull was then exposed, 2% lidocaine was applied to thesite of incision, and a small brass tube was secured to the skullsurface along the sagittal suture at bregma with vet bond and adheredwith dental cement. Mice were given a recovery period of 24-48 hoursbefore beginning the experimental sessions.

Drug Administration.

The peptide was prepared from a 10 mM aqueous stock solution and diluteddown to 10 μM. Topical administration of either peptide or paxillineconsisted of direct application of 1 μL solution to the exposed surfaceof the inferior colliculus at concentration dosages of 10 μM or an i.p.injection at a dose of 10 which is 0.33 ng/mg body weight for a 30 gmouse. Fresh solutions were made prior to each experiment.

Auditory Brainstem Response Procedures.

ABR recordings were acquired after the mice were anesthetized withketamine (120 mg/kg) and xylazine (10 mg/kg) i.p., and respiration wasmonitored throughout to determine when additional supplemental doseswere needed. Body temperature was kept constant at 37° C. using afeedback controlled heating pad (Physitemp TCAT2-LV Controller, Clifton,N.J.). Stimuli and recordings were generated digitally and controlledusing a TDT RZ6 Multi-I/O Processor and their BioSig/SigGen software.Acoustic signals were played through a multi-field (MF1) magneticspeaker (TDT, Alachua, Fla.) with a total harmonic distortion<=1% from 1kHz to 50 kHz, centered 0° azimuth in regards to the animal at adistance of 10 cm from the ear pinna. Tone bursts were presented atfrequencies of 6, 12, 16, 20, 24, and 36 kHz (3 ms duration, 1 msrise/fall time, alternating polarity) at a rate of 29 per second,attenuated in 5 dB steps from 80 dB SPL to 15 dB below threshold or 5 dBSPL, whichever was lower. Threshold was determined by visual inspectionas the lowest intensity level which produced a defined wave in bothreplicates. All signals were calibrated using a Larsen Davispreamplifier, model 2221, with a ¼″ microphone and a Larson Davis CAL200Precision Acoustic Calibrator (PCB Piezotronics, Inc., Depew, N.Y.). ABRrecordings were acquired using a TDT RA4LI low-impedance digitalheadstage and RA4PA Medusa preamp with the active (noninverting)electrode inserted at the vertex, the reference (inverting) electrodebelow the left ear, and the ground electrode below the right ear. Theresponses were amplified (20×), filtered (300 Hz-3 kHz), and averagedusing BioSig software and the System III hardware (TDT) data-acquisitionsystem. A total of 256 tone burst signal and 150 GIN signal recordingswere replicated for each acquisition, and muscle artifacts exceeding 7uV were rejected from the averaged response. All recordings took placein a soundproof booth lined with echo-attenuating acoustic foam. ABRwaveforms were analyzed using a custom MatLab program that automaticallydetermined peak latencies and amplitudes in combination with secondaryverification by an experimenter blind to the treatment group.

Extracellular Recording Procedures.

The right IC was stereotaxically located⁷⁴ and exposed via a small (<1.0mm) craniotomy. Prior to recording, chlorprothixene (Taractin®, 5-12μl/g i.m.) was administered. The animal was then secured in a customstereotaxic frame (Newport-Klinger) that was located in a heated (34°C.) chamber lined with sound-absorbing foam (Sonex®). Multi-unitextracellular activity was recorded using vertically oriented singleshank silicon acute penetrating 16-channel electrodes with an impedanceranging from 1.2 to 2.1 MΩ (Type-A, 3 mm×100 μm; NeuroNexusTechnologies). The electrode was positioned stereotaxically over the ICafter reference to the lambda landmark on the skull, and was advanceddorsoventrally into the IC by a micro positioner (Newport-Klinger PMC100). The output from the electrode was attached to a low noise (5-6 uVnoise floor) preamplifier (RA16), having an operating range of ±7 mV.Neural events were acquired and visualized in real-time using the OpenExsoftware platform (TDT, Inc.) and a custom designed MATLAB® (TheMathWorks, Inc., Matick, Mass.) graphical interface. Neural recordingsfrom each channel were then filtered (300-3000 Hz), amplified, andsampled at 25 kHz in a 1.25 ms time window subsequent to the eventcrossing a voltage discriminator. A spike triggering threshold of 4:1signal to noise ratio (SNR) was automatically set for all channels. Thesearch signal used to estimate the spike triggering thresholds was a50-ms broadband noise stimulus presented at 60 dB SPL at a rate of 5/s.Each penetration typically yielded 8-16 active channels. Recordingsessions lasted an average of 6-8 hours, and if at any time a mouseshowed signs of discomfort, like excessive movement, it was removed fromthe apparatus and testing was halted.

Stimulus generation and presentation. Noise and tone bursts weregenerated digitally (Real-time Processor Visual Design Studio (RVPds),TDT) using a System 3 processor and D/A converter (TDT RX6) with 200 kHzsampling rate. The signals were routed to an electrostatic speaker (TDTES1) with a flat frequency response from 4 to 110 kHz. This speaker wasplaced at 60° azimuth contralateral to the recording site. Harmonicdistortions were measured with a Dynamic Signal Analyzer (HP 35665A) andwere at least 60 dB below the primary signal. The distance between thespeaker and the pinna was fixed at 22.5 cm and calibrated using a B&K2610 amplifier and a ¼″ microphone placed at the location of the pinna.eRFs from all active channel were acquired simultaneously using 25 ms (5ms rise/fall) tone burst stimuli presented from 0 to 90 dB SPL in 5 dBsteps and from 2 to 64 kHz for a total of 2125 frequency and intensitycombinations that were presented pseudo-randomly five times at a rate of10/s.

Spike Sorting.

Spike waveforms were processed in MATLAB® using the TDT OpenDeveloperActiveX controls and passed to AutoClass C v3.3.4, an unsupervisedBayesian classification system that seeks a maximum posteriorprobability classification, developed at the NASA Ames ResearchCenter^(75,76). AutoClass scans the dataset of voltage-time waveformsaccording to custom specified spike parameters to produce the best fitclassifications of the data, which may include distinct single- andmulti-unit events, as well as noise. To discriminate the signal fromnoise in the present study, the variance of the background noise wasestimated as the quartile range of the first five digitization points ofthe spike waveform, at these are recorded prior to thethreshold-crossing event. To avoid overloading AutoClass with excessivenoise, which leads to over-classification, this noise measure is used toscreen the event waveform data, such that only voltage points withabsolute values greater than this noise floor were presented for use inthe classification. Once the classes had been determined in each channelof data, they were visualized within a custom MATLAB® program andassigned to multi-unit, single-unit, or noise classes. Event classeswhich were categorized as noise were subsequently discarded, and unitswith distinct biphasic waveforms and good SNR were classified assingle-units. As most channels recorded information elicited from thespiking of two or more neurons, all recordings units in this paper wereconsidered to be MUA⁷⁷. Nonetheless, there was no observation of anyconsistent differences in the RFs between single units and multi-unitclusters.

Data Analyses.

FRAs were analyzed using a custom MATLAB® program We classified RFtuning using a method similar to that used to classify neurons in theprimary AC⁷⁸. The frequency at which driven activity is responsive atthe lowest intensity (threshold) is classified as the characteristicfrequency (CF) and the point in the receptive field, which elicits themaximal driven activity is categorized as the best frequency (BF). Acustom MATLAB® program was used to calculate the edges of each eRF, andthis was verified via visual inspection to ensure no non-driven activitywas included in the calculation. The edges of the RF were defined, in 10dB steps above threshold, as the activity levels that were equal to orgreater than the background rate and at least 15% of the maximum rate.Each RF was categorized into low-BF (<15 kHz), mid-BF (15-30 kHz), andhigh-BF (>30 kHz) groups based on the topographical representationproposed by Willott⁷⁹. The maximum driven rate, as well as the totalspike counts, were taken from the baseline.

Statistical analysis and graphs were created using GraphPad Prismversion 6.01 for Windows (GraphPad Software, La Jolla, Calif.). Themajority of the data results are presented using box plots, which allowsfor both mean, along with the ±standard error of the mean (SEM) andmedian values to be denoted. A one-way ANOVA test and Tukey's repeatedmeasures analysis procedure were used to evaluate the effects of thepeptide on the spike counts within the baseline eRF. Alpha was set at0.05 for all statistical tests.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—LS3 Peptide Suppresses BK Channel Activity and DecreasesSpontaneous and Sound-Evoked Activity of Inferior Colliculus Neurons

The inventors propose that BK channel-directed peptides can be used toincrease temporal precision and/or gain in central auditory processingin order to alleviate some of the deficits caused by age-related hearingloss.

The inventors have identified a novel BK channel directed peptide (LS3;SEQ ID NO:3) that suppresses BK channel via modulation of the alphasubunit activity, rather than blocking the pore to prevent ion flow.This peptide is 9 amino acids in length (sequence: CRRGLVQVC; SEQ IDNO:3) with an N- and C-terminal disulfide bridge. Application of thispeptide at 500 pM-500 nM to BK channels expressed in HEK293 cells causesa strong suppression of channel openings (by ˜⅔rds at 60 mV). Little tono suppression is seen when applied at 50 pM, indicating an IC50 in thelow hundreds of pM range.

To provide a measure of whether the peptide modulates BK channelfunction specifically, the inventors tested the peptide for the abilityto alter a BK channel-mediated behavior in Caenorhabditis elegans. C.elegans express a wide variety of highly conserved potassium channels,in addition to about two-thirds of all mammalian proteins. The BKchannel is highly conserved between worms and mammals. This assayrevealed that the peptide reduces crawl speed in wild-type but not BKchannel-null worms. Moreover, the reduction in crawl speed can berescued in the BK channel-null worms with expression of either thewild-type worm BK channel or the wild-type human BK channel. These dataindicate that the peptide acts with a high level of specificity at boththe nematode and mammalian BK channel.

This novel BK channel modulating peptide shows the ability to cross themammalian blood-brain barrier as well as alter neuronal activity invivo. When applied to the surface of the dura in mouse, this peptidealters neuronal properties of inferior colliculus neurons after about 10minutes, showing that the peptide can cross the blood-brain barrier(BBB). To further demonstrate that the peptide can cross the blood-brainbarrier (BBB), the inventors attached a fluorophore to the peptide. Whenthis compound was applied to the dura, fluorescence could be seen at˜1200 microns in depth after 8 hours. This indicates that not only canthe peptide cross the BBB, but it can also port other molecules acrossas well.

Once across the blood-brain barrier, the peptide shows aconcentration-dependent effect on sound-evoked activity among inferiorcolliculus neurons. When applied at 10 microM (2 uL), the peptidedecreases spontaneous and sound-evoked activity of inferior colliculusneurons by suppressing pre-synaptic input for neurons of the centralnucleus of the inferior colliculus. These results vary among thetonotopic regions of the inferior colliculus with the most robustdecreases seen in the most ventral locations. This suppression ofactivity is similar to the strong suppression of activity seen with aknown BK channel blocker, paxilline. These similarities between theeffect of peptide and paxilline suggest that the peptide is alteringinferior colliculus neuronal properties through a BK channel-mediatedmechanism.

In contrast, when applied at 1 microM, the pre-synaptic suppressionappears to be reduced enough to reveal interesting post-synapticmodifications. This is particularly true for dorsal units, for which thepre-synaptic suppression of activity is less. At this concentration, thepeptide lowers sound-evoked thresholds and provides better definition toneuronal receptive fields in aged mice. The peptide also suppressesspontaneous activity. This improvement in sound-evoked thresholds andreceptive field definition in aged mice is clinically relevant to theimprovement of age-related hearing loss.

This peptide resulted from a screen that yielded other BK channelmodulating peptides as well. These peptides are all 9 amino acids inlength with a C-to-N terminal disulfide bond. Some of the other peptidesshare motifs with the peptide discussed above. Putatively, these motifsshare functional qualities. For example, both this and another peptide(LS1), which share some but not all motifs, are able to modulatephosphorylation at a specific site on the BK channel. Data suggests thatLS1 also is able to cross the blood-brain barrier and alter neuronalproperties in vivo in a similar manner to the peptide discussed above.Pre-synaptic suppression of activity is not as strong for LS1 as LS3,which may prove to be advantageous. Results are shown in the Figures.Scott L L et al., “A novel BK channel-targeted peptide suppresses soundevoked activity in the mouse inferior colliculus,” Sci Rep, 2017 Feb.14, 7:42433, is incorporated herein by reference in its entirety,including all Figures and Results.

The present inventors have identified a cohort of peptides that modulatethe large conductance calcium-activated potassium channel. Onewell-characterized peptide, LS3, reduces opening of the BK channel. TheBK channel is gated both by voltage and intracellular calcium. It iswidely expressed in human tissue where it regulates smooth muscle tone,endocrine secretion and neuronal excitability. While widely expressed,global knockdown of the constitutive pore-forming a subunit orregulatory subunits is not lethal.

The BK channel is considered a promising pharmacological target for abroad range of diseases. As outlined above, the peptide LS3 showspromise for treating age-related hearing loss. This peptide and othersin the cohort have other potential uses as well. Our data most obviouslypoints to an additional use in treating epilepsy. A BK channelgain-of-function mutation is associated with an increased risk forepilepsy in humans and animal models. Pharmacological treatment with aBK channel blocker is effective in some in vivo and in vitro seizuremodels. More specifically, a BK channel blocker can reduce audiogenicseizure activity in a mouse epilepsy model. The inferior colliculus is acritical site for the initiation of audiogenic seizures. The action ofLS3 as a BK channel closer predicts similar activity in epilepsy modelsto known BK channel blockers. The reduction in activity in the inferiorcolliculus, as shown at the higher dose, specifically predicts aprotective action against audiogenic seizures.

There is also evidence that LS3 and other peptides in this cohort(Table 1) may be useful in addressing alcohol addiction. InCaenorhabditis elegans and Drosophila, null mutations in the highlyconserved invertebrate BKα channels restrict acute ethanol intoxicationand tolerance. These invertebrates are intoxicated by similar internalconcentrations of alcohol as mammals and likely share similar mechanismsof intoxication, tolerance and other alcohol-related behaviors. LS3 andanother peptide in this cohort reduce acute alcohol intoxication in C.elegans. Other peptides in this cohort also reduce behavioral symptomsof withdrawal from chronic alcohol exposure in C. elegans. The inventorspropose that these peptides would influence alcohol behaviors inmammals.

LS3 may also have therapeutic value in treating cancers. BK channels areexpressed in human tumor cells where, in many cases, they support tumorgrowth and spreading. Antagonists stop tumor proliferation in vitro. Apreliminary experiment suggests LS3 can disrupt cell proliferation.

In addition, the peptides may have therapeutic value in treatinglate-stage neurodegenerative diseases. BK channel blockers in thecentral nervous system (CNS) enhance synaptic transmission, potentiallyrestoring some cognitive function in chronic neurodegenerative diseases(e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington'sdisease). Moreover, BK channel blockers can modulate microgliaactivation, which could be protective in later stages of these samechronic neurodegenerative diseases.

By virtue of their receptor binding ability, the peptides may also beused as tools for research and drug screening, for example, incompetitive assays to identify and compare other agents that havereceptor binding affinity. Optionally, the peptides may include adetectable label for use in this way.

LS3 could be used to modulate circadian rhythms. Changes in BK channelexpression and composition in the suprachiasmatic nucleus (SCN) in turnalters circadian behavior and SCN neuronal firing rates. It is knownthat BK currents are larger during the night than during the day, andinactivation of the BK channel at night can switch SCN activity todaytime levels. LS3 is likely to provide similar regulation of SCNactivity.

It is known that knockdown of the a subunit of the BK channel causesdefects in motor performance, erectile dysfunction and over-activebladders in mouse. Experiments in ex vivo preparations of bladdersuggest that LS3 does not show typical anti-BK channel activity insmooth muscle. This is fortuitous for reducing negative side effects inthe above applications.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

We claim:
 1. A method for treating presbycusis in a subject in needthereof, the method comprising: administering an effective amount of apeptide, wherein the presbycusis is associated with aberrant neuralactivity that is modulated by large conductance Ca2+ activated K+ (BK)channel function in one or more cells of the subject; and wherein theamino acid sequence of the peptide comprises CRRGLVQVC (SEQ ID NO:3),and wherein the peptide is a circular peptide having a disulfide bridgebetween cysteines.
 2. The method of claim 1, wherein the peptide isadministered to the subject by one or more routes selected fromintravascular, intramuscular, subcutaneous, oral, intranasal,intra-ocular, topical, and transdermal.
 3. The method of claim 2,wherein the subject is a human.
 4. The method of claim 1, wherein theamino acid sequence of the peptide consists of: CRRGLVQVC (SEQ ID NO:3).5. A method comprising: modulating, in vitro, large conductance Ca2+activated K+ (BK) channel activity in a mammalian cell having a BKchannel, comprising contacting the cell with a peptide, wherein theamino acid sequence of the peptide comprises CRRGLVQVC (SEQ ID NO:3),wherein the peptide is a circular peptide having a disulfide bridgebetween cysteines.
 6. The method of claim 5, wherein the peptidecomprises a detectable label.
 7. The method of claim 5, wherein theamino acid sequence of the peptide consists of CRRGLVQVC (SEQ ID NO:3).8. A method for delivering a cargo moiety to the brain of a subject,through the blood-brain barrier (BBB), the method comprising:administering via a route selected from one or more of intravascular,intramuscular, subcutaneous, oral, intranasal, intra-ocular, topical,and transdermal, an effective amount of a polypeptide construct to thesubject, thereby delivering the cargo moiety to the brain of thesubject, wherein the polypeptide construct comprises a peptideconjugated to the cargo moiety; wherein the amino acid sequence of thepeptide comprises: CRRGLVQVC (SEQ ID NO:3), wherein the peptide is acircular peptide having a disulfide bridge between cysteines; whereinthe peptide and the cargo are delivered to the brain of the subject. 9.The method of claim 8, wherein the cargo moiety comprises a detectablelabel.
 10. The method of claim 8, wherein the amino acid sequence of thepeptide consists of CRRGLVQVC (SEQ ID NO: 3).